HEN1 is the Arabidopsis SAM-dependent small RNA 3' terminal 2'-O-methyltransferase. It methylates the 2'-OH of the 3'-terminal ribose on miRNA/miRNA-star and siRNA/siRNA-star duplexes after Dicer-like processing, protecting small RNAs from uridylation, trimming, and degradation. HEN1 acts in the DCL1/HYL1-associated small-RNA maturation context and supports miRNA, siRNA, ta-siRNA, and antiviral silencing outputs; floral and leaf patterning phenotypes are downstream consequences of destabilized small RNAs.
Definition: A small-RNA metabolic process in which methylation of the 2-prime-OH of the 3-prime-terminal ribose protects mature small RNAs from uridylation, trimming, and exonucleolytic decay.
Justification: HEN1's direct molecular function is captured by small RNA 2-prime-O-ribose methyltransferase activity, but the current core-function summary also needs to distinguish the downstream protective effect of that modification. A process term for small-RNA 3-prime-end protection would avoid reusing the same catalytic molecular-function term for both methyl transfer and stabilization.
Parent term: RNA processing
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
|
GO:0034587
piRNA processing
|
IBA
GO_REF:0000033 |
REMOVE |
Summary: HEN1-family proteins include animal piRNA methyltransferases, but Arabidopsis HEN1 methylates plant miRNA and siRNA duplexes.
Reason: piRNA processing is an over-transfer from metazoan HEN1/HENMT1 family context and is not supported for Arabidopsis HEN1.
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GO:0008171
O-methyltransferase activity
|
IBA
GO_REF:0000033 |
MODIFY |
Summary: HEN1 is an RNA methyltransferase, but O-methyltransferase activity is less specific than the small-RNA 2-prime-O-ribose methyltransferase term.
Reason: Use the small-RNA terminal 2-prime-O-methyltransferase term to capture HEN1 substrate and reaction specificity.
Proposed replacements:
small RNA 2'-O-ribose methyltransferase activity
|
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GO:0003755
peptidyl-prolyl cis-trans isomerase activity
|
IEA
GO_REF:0000002 |
REMOVE |
Summary: HEN1 includes a PPIase-like/FKBP-like domain, but its curated function is small-RNA methyltransferase activity.
Reason: The PPIase-like/FKBP-like domain is present, but no evidence supports HEN1 peptidyl-prolyl isomerase catalytic activity; the supported biochemical function is small-RNA methyltransferase activity.
|
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GO:0005634
nucleus
|
IEA
GO_REF:0000044 |
ACCEPT |
Summary: HEN1 is associated with the nuclear DCL1/HYL1 small-RNA maturation context.
Reason: The best-supported site for HEN1 action is nuclear small-RNA processing/methylation before or around AGO loading.
Supporting Evidence:
file:ARATH/HEN1/HEN1-deep-research-falcon.md
Direct interaction mapping and mechanistic models place HEN1 in the nuclear miRNA-processing context.
|
|
GO:0008171
O-methyltransferase activity
|
IEA
GO_REF:0000002 |
MODIFY |
Summary: HEN1 is an RNA methyltransferase, but O-methyltransferase activity is less specific than the small-RNA 2-prime-O-ribose methyltransferase term.
Reason: Use the small-RNA terminal 2-prime-O-methyltransferase term to capture HEN1 substrate and reaction specificity.
Proposed replacements:
small RNA 2'-O-ribose methyltransferase activity
|
|
GO:0008173
RNA methyltransferase activity
|
IEA
GO_REF:0000002 |
MODIFY |
Summary: HEN1 is an RNA methyltransferase, but RNA methyltransferase activity is less specific than the small-RNA 2-prime-O-ribose methyltransferase term.
Reason: Use the small-RNA terminal 2-prime-O-methyltransferase term to capture HEN1 substrate and reaction specificity.
Proposed replacements:
small RNA 2'-O-ribose methyltransferase activity
|
|
GO:0016070
RNA metabolic process
|
IEA
GO_REF:0000117 |
MODIFY |
Summary: HEN1 participates in small-RNA metabolism by methylating mature miRNA/siRNA duplexes.
Reason: RNA metabolic process is too broad; small-RNA methylation and miRNA/siRNA processing-stabilization outputs are more informative.
Proposed replacements:
small RNA 2'-O-ribose methyltransferase activity
miRNA processing
|
|
GO:0090486
small RNA 2'-O-ribose methyltransferase activity
|
IEA
GO_REF:0000003 |
ACCEPT |
Summary: HEN1 catalyzes terminal 2-prime-O-ribose methylation of plant small RNAs. The HEN1 crystal structure (PDB 3HTX) shows the SAH cofactor product bound in the Rossmann MTase domain as in classical SAM-dependent methyltransferases, with a catalytic Mg ion coordinating the 3-prime-terminal 2-prime and 3-prime hydroxyls, consistent with 2-prime-O-ribose methyl transfer.
Reason: This is the most specific molecular-function term for Arabidopsis HEN1.
Supporting Evidence:
file:ARATH/HEN1/HEN1-deep-research-falcon.md
HEN1 is a small RNA 3-prime-terminal 2-prime-O-methyltransferase.
file:ARATH/HEN1/HEN1-uniprot.txt
Reaction=small RNA 3'-end nucleotide + S-adenosyl-L-methionine = small RNA 3'-end 2'-O-methylnucleotide + S-adenosyl-L-homocysteine + H(+);
file:interpro/panther/PTHR21404/PTHR21404-entries.csv
Q9C5Q8,Small RNA 2'-O-methyltransferase
PMID:19812675
The MTase domain of HEN1 adopts a core α/β Rossmann structure, in which the cofactor product AdoHcy is bound as in classical S-adenosyl-l-methionine (AdoMet)-dependent MTases
PMID:19812675
both the 2′ and 3′ hydroxyls of G22m and the side chains of four invariant residues (E796, E799, H800 and H860) are coordinated to a metal ion, Mg2+
|
|
GO:0090486
small RNA 2'-O-ribose methyltransferase activity
|
IEA
GO_REF:0000116 |
ACCEPT |
Summary: HEN1 catalyzes terminal 2-prime-O-ribose methylation of plant small RNAs. The 3.1 A crystal structure of full-length Arabidopsis HEN1 (PDB 3HTX) bound to a 22-nucleotide small RNA duplex with the cofactor product SAH (S-adenosyl-L-homocysteine) and Mg directly visualizes the substrate as a small-RNA duplex and confirms terminal 2-prime-O-methyltransferase chemistry.
Reason: This is the most specific molecular-function term for Arabidopsis HEN1.
Supporting Evidence:
file:ARATH/HEN1/HEN1-deep-research-falcon.md
HEN1 is a small RNA 3-prime-terminal 2-prime-O-methyltransferase.
file:ARATH/HEN1/HEN1-uniprot.txt
Reaction=small RNA 3'-end nucleotide + S-adenosyl-L-methionine = small RNA 3'-end 2'-O-methylnucleotide + S-adenosyl-L-homocysteine + H(+);
file:interpro/panther/PTHR21404/PTHR21404-entries.csv
Q9C5Q8,Small RNA 2'-O-methyltransferase
PMID:19812675
report the 3.1 A crystal structure of full-length HEN1 from Arabidopsis in complex with a 22-nucleotide small RNA duplex and cofactor product S-adenosyl-l-homocysteine
PMID:19812675
A subset of small RNAs, such as microRNAs and small interfering RNAs (siRNAs) in plants, Piwi-interacting RNAs in animals and siRNAs in Drosophila, requires an additional crucial step for their maturation; that is, 2'-O-methylation on the 3' terminal nucleotide. A conserved S-adenosyl-l-methionine-dependent RNA methyltransferase, HUA ENHANCER 1 (HEN1), and its homologues are responsible for this specific modification.
|
|
GO:0010267
ta-siRNA processing
|
IMP
PMID:15469823 Endogenous trans-acting siRNAs regulate the accumulation of ... |
KEEP AS NON CORE |
Summary: HEN1 supports ta-siRNA processing by methylating and stabilizing the small-RNA duplexes that feed these pathways.
Reason: HEN1 is required for stable miRNA/siRNA pathway outputs, but the direct catalytic step is small-RNA 2-prime-O-methylation rather than Dicer cleavage or AGO-mediated target repression.
Supporting Evidence:
file:ARATH/HEN1/HEN1-uniprot.txt
Can methylate 3'-end of microRNAs (miRNAs), small interfering RNAs (siRNas) and trans-acting small interfering RNAs (ta-siRNAs).
|
|
GO:0005737
cytoplasm
|
ISM
GO_REF:0000122 |
UNDECIDED |
Summary: The AtSubP-inferred cytoplasm row lacks positive experimental support for HEN1 cytoplasmic localization.
Reason: HEN1's best-supported context is small-RNA methylation around nuclear miRNA/siRNA maturation; downstream cytoplasmic small-RNA turnover is not sufficient evidence for a cytoplasm localization annotation.
|
|
GO:0010305
leaf vascular tissue pattern formation
|
IMP
PMID:22623415 The microRNA pathway genes AGO1, HEN1 and HYL1 participate i... |
KEEP AS NON CORE |
Summary: hen1 mutants affect leaf vascular tissue pattern formation.
Reason: The developmental process is a downstream phenotype of altered miRNA/siRNA stability rather than a direct HEN1 function.
|
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GO:0010589
leaf proximal/distal pattern formation
|
IMP
PMID:22623415 The microRNA pathway genes AGO1, HEN1 and HYL1 participate i... |
KEEP AS NON CORE |
Summary: hen1 mutants affect leaf proximal/distal pattern formation.
Reason: The developmental process is a downstream phenotype of altered miRNA/siRNA stability rather than a direct HEN1 function.
|
|
GO:0009616
RNAi-mediated antiviral immune response
|
IMP
PMID:17090584 Four plant Dicers mediate viral small RNA biogenesis and DNA... |
KEEP AS NON CORE |
Summary: HEN1 contributes to virus-induced silencing and viral-small-RNA stability through terminal small-RNA methylation.
Reason: The antiviral role is a pathway output of HEN1-mediated small-RNA stabilization; the direct catalytic function remains small-RNA 2-prime-O-methylation rather than Dicing or Argonaute effector activity.
Supporting Evidence:
PMID:17090584
DCL4 in conjunction with RDR6 and HEN1 specifically facilitates extensive virus-induced silencing in new growth.
|
|
GO:0005634
nucleus
|
IDA
PMID:17442570 Identification of nuclear dicing bodies containing proteins ... |
ACCEPT |
Summary: The cited D-body paper places plant miRNA processing in the nucleus but directly images DCL1/HYL1/SE rather than HEN1.
Reason: Although PMID:17442570 directly images other D-body proteins, HEN1 has independent support for the same nuclear miRNA-processing context, so the nucleus annotation is retained consistently.
Supporting Evidence:
file:ARATH/HEN1/HEN1-deep-research-falcon.md
Direct interaction mapping and mechanistic models place HEN1 in the nuclear miRNA-processing context.
|
|
GO:0005737
cytoplasm
|
IDA
PMID:17442570 Identification of nuclear dicing bodies containing proteins ... |
UNDECIDED |
Summary: The cited D-body paper contrasts animal cytoplasmic miRNA processing with plant nuclear processing and does not directly support HEN1 cytoplasmic localization.
Reason: HEN1 may influence cytoplasmic small-RNA turnover indirectly, but PMID:17442570 does not provide direct HEN1 cytoplasm evidence.
|
|
GO:0008173
RNA methyltransferase activity
|
IDA
PMID:15705854 Methylation as a crucial step in plant microRNA biogenesis. |
MODIFY |
Summary: Experimental Arabidopsis work supports HEN1-dependent methylation/protection of miRNAs and siRNAs. The HEN1 crystal structure (PDB 3HTX) confirms it is a SAM-dependent RNA methyltransferase, with the AdoHcy (SAH) product bound in a Rossmann MTase domain as in classical AdoMet-dependent MTases.
Reason: Use the small-RNA terminal 2-prime-O-methyltransferase term to capture HEN1 substrate and reaction specificity.
Proposed replacements:
small RNA 2'-O-ribose methyltransferase activity
Supporting Evidence:
file:ARATH/HEN1/HEN1-deep-research-falcon.md
HEN1 catalyzes transfer of a methyl group from AdoMet to the 2-prime-OH of the 3-prime-terminal nucleotide of small RNAs.
file:ARATH/HEN1/HEN1-uniprot.txt
Methyltransferase that adds a methyl group to the ribose of the last nucleotide of small RNAs (sRNAs).
PMID:19812675
A conserved S-adenosyl-l-methionine-dependent RNA methyltransferase, HUA ENHANCER 1 (HEN1), and its homologues are responsible for this specific modification.
PMID:19812675
The MTase domain of HEN1 adopts a core α/β Rossmann structure, in which the cofactor product AdoHcy is bound as in classical S-adenosyl-l-methionine (AdoMet)-dependent MTases
|
|
GO:0008173
RNA methyltransferase activity
|
IMP
PMID:16111943 Methylation protects miRNAs and siRNAs from a 3'-end uridyla... |
MODIFY |
Summary: Experimental Arabidopsis work supports HEN1-dependent methylation/protection of miRNAs and siRNAs. The HEN1 crystal structure (PDB 3HTX) shows a catalytic Mg2+ ion coordinated by the 2-prime and 3-prime hydroxyls of the 3-prime-terminal nucleotide and four invariant active-site residues, defining a Mg2+-dependent 2-prime-O-methylation mechanism.
Reason: Use the small-RNA terminal 2-prime-O-methyltransferase term to capture HEN1 substrate and reaction specificity.
Proposed replacements:
small RNA 2'-O-ribose methyltransferase activity
Supporting Evidence:
file:ARATH/HEN1/HEN1-deep-research-falcon.md
HEN1 catalyzes transfer of a methyl group from AdoMet to the 2-prime-OH of the 3-prime-terminal nucleotide of small RNAs.
file:ARATH/HEN1/HEN1-uniprot.txt
Methyltransferase that adds a methyl group to the ribose of the last nucleotide of small RNAs (sRNAs).
PMID:19812675
Metal ion coordination by both 2' and 3' hydroxyls on the 3'-terminal nucleotide and four invariant residues in the active site of the methyltransferase domain suggests a novel Mg(2+)-dependent 2'-O-methylation mechanism.
PMID:19812675
both the 2′ and 3′ hydroxyls of G22m and the side chains of four invariant residues (E796, E799, H800 and H860) are coordinated to a metal ion, Mg2+
|
|
GO:0035279
miRNA-mediated gene silencing by mRNA destabilization
|
IMP
PMID:15851028 microRNA-directed phasing during trans-acting siRNA biogenes... |
KEEP AS NON CORE |
Summary: HEN1 supports miRNA-mediated gene silencing by mRNA destabilization by methylating and stabilizing the small-RNA duplexes that feed these pathways.
Reason: HEN1 is required for stable miRNA/siRNA pathway outputs, but the direct catalytic step is small-RNA 2-prime-O-methylation rather than Dicer cleavage or AGO-mediated target repression.
Supporting Evidence:
file:ARATH/HEN1/HEN1-uniprot.txt
Can methylate 3'-end of microRNAs (miRNAs), small interfering RNAs (siRNas) and trans-acting small interfering RNAs (ta-siRNAs).
|
|
GO:0035196
miRNA processing
|
IMP
PMID:12747833 Arabidopsis HEN1: a genetic link between endogenous miRNA co... |
ACCEPT |
Summary: HEN1 supports miRNA processing by methylating and stabilizing the small-RNA duplexes that feed these pathways.
Reason: HEN1 methylation is a required post-dicing maturation/protection step in plant miRNA biogenesis, so miRNA processing is supported even though the more specific catalytic role is small-RNA 2-prime-O-methylation.
Supporting Evidence:
file:ARATH/HEN1/HEN1-uniprot.txt
Can methylate 3'-end of microRNAs (miRNAs), small interfering RNAs (siRNas) and trans-acting small interfering RNAs (ta-siRNAs).
file:ARATH/HEN1/HEN1-deep-research-falcon.md
HEN1 methylation is a defining post-dicing step in plants that stabilizes miRNAs and siRNAs.
PMID:19812675
A subset of small RNAs, such as microRNAs and small interfering RNAs (siRNAs) in plants, Piwi-interacting RNAs in animals and siRNAs in Drosophila, requires an additional crucial step for their maturation; that is, 2'-O-methylation on the 3' terminal nucleotide.
|
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GO:0009909
regulation of flower development
|
IMP
PMID:11917084 Formation of corymb-like inflorescences due to delay in bolt... |
KEEP AS NON CORE |
Summary: hen1 mutants affect regulation of flower development.
Reason: The developmental process is a downstream phenotype of altered miRNA/siRNA stability rather than a direct HEN1 function.
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GO:0010093
specification of floral organ identity
|
IMP
PMID:11874905 HEN1 functions pleiotropically in Arabidopsis development an... |
KEEP AS NON CORE |
Summary: hen1 mutants affect specification of floral organ identity.
Reason: The developmental process is a downstream phenotype of altered miRNA/siRNA stability rather than a direct HEN1 function.
|
Q: Should piRNA-processing IBA transfer be excluded systematically for plant HEN1 proteins without piRNA pathway evidence?
Q: Should GO add a process term such as small RNA 3-prime-end protection by 2-prime-O-methylation for HEN1-dependent protection from uridylation, trimming, and decay?
Experiment: Quantify methylation efficiency of Arabidopsis HEN1 on defined miRNA, siRNA, and ta-siRNA duplex substrates with varying overhang geometry.
Experiment: Map the compartment and timing of HEN1 methylation relative to DCL1/HYL1 processing and AGO1 loading using endogenous tagged proteins.
The research report should be a detailed narrative explaining the function, biological processes, and localization of the gene product. Citations should be given for all claims.
You should prioritize authoritative reviews and primary scientific literature when conducting research. You can supplement
this with annotations you find in gene/protein databases, but these can be outdated or inaccurate.
We are specifically interested in the primary function of the gene - for enzymes, what reaction is catalyzed, and what is the substrate specificity? For transporters, what is the substrate? For structural proteins or adapters, what is the broader structural role? For signaling molecules, what is the role in the pathway.
We are interested in where in or outside the cell the gene product carries out its function.
We are also interested in the signaling or biochemical pathways in which the gene functions. We are less interested in broad pleiotropic effects, except where these elucidate the precise role.
Include evidence where possible. We are interested in both experimental evidence as well as inference from structure, evolution, or bioinformatic analysis. Precise studies should be prioritized over high-throughput, where available.
The research target matches the UniProt-provided identity: Arabidopsis thaliana HEN1 (HUA ENHANCER 1) is a small RNA 3′-terminal 2′-O-methyltransferase (EC 2.1.1.386) that uses S-adenosyl-L-methionine (SAM/AdoMet) to methylate the 2′-OH of the 3′-terminal ribose of plant small RNAs. Structural and biochemical work confirms the multidomain architecture expected for plant HEN1, including two dsRNA-binding domains (dsRBDs) and a La-motif-containing domain, plus a C-terminal Rossmann-like methyltransferase domain. (huang2009structuralinsightsinto pages 1-2, huang2009structuralinsightsinto pages 2-4, ji2012regulationofsmall pages 1-3)
Plant HEN1 is distinct from the metazoan homolog HENMT1 (primarily piRNA methyltransferase) and from bacterial Hen1 proteins (different domain architecture and biological context), avoiding symbol ambiguity. (peng2018identificationofsubstrates pages 1-2, ji2012regulationofsmall pages 1-3)
In plants, miRNAs and many siRNAs acquire a 2′-O-methyl group at the 3′ terminal nucleotide. This is a post-processing stabilization step that is central to small RNA metabolism because it alters susceptibility to 3′ end modification and degradation. (ji2012regulationofsmall pages 1-3, ding2023micrornaproductionin pages 1-2)
HEN1 catalyzes transfer of a methyl group from AdoMet to the 2′-OH of the 3′-terminal nucleotide of small RNAs; AdoHcy is produced as the cofactor product. Importantly, plant HEN1 methylates both strands of the miRNA/miRNA or siRNA/siRNA duplex (in succession) rather than only a single guide strand. (baranauske2015functionalmappingof pages 1-2, ji2012regulationofsmall pages 3-4, huang2009structuralinsightsinto pages 1-2)
Substrates: HEN1 acts on 21–24 nt small RNA duplexes (miRNA/miRNA and siRNA/siRNA) with the typical 2-nt 3′ overhangs generated by Dicer-like processing. (ji2012regulationofsmall pages 3-4, huang2009structuralinsightsinto pages 1-2)
Specificity: HEN1 is broadly non-sequence-specific; recognition is driven by RNA duplex geometry and terminal features (overhangs and ribose hydroxyls) rather than nucleobases. (huang2009structuralinsightsinto pages 2-4, kaldis2024molecularbasisof pages 11-12)
A landmark structure solved the full-length Arabidopsis HEN1 in complex with a 22-nt RNA duplex and AdoHcy at 3.1 Å resolution, showing that HEN1 binds the duplex as a monomer and uses multiple RNA-binding modules to measure and engage duplex ends. The La-motif-containing domain recognizes one duplex terminus/overhang, while the methylated strand’s 3′-terminal nucleotide is positioned into the MTase active site. (huang2009structuralinsightsinto pages 1-2, huang2009structuralinsightsinto pages 2-4)
The retrieved structural figure provides direct visual evidence for these domain arrangements and RNA-end recognition principles. (huang2009structuralinsightsinto media 62c0a606, huang2009structuralinsightsinto media 867c9cf3)
A recent mechanistic analysis reports kinetic parameters for full-length plant HEN1 of approximately KRNA ≈ 0.22 mM, KSAM ≈ 1.7 mM, and kcat ≈ 3.0 min−1, and supports a model in which active-site chemistry involves conserved residues coordinating a divalent metal and engaging the 2′/3′ hydroxyls at the terminal ribose. This work also reinforces that the N-terminal region enhances substrate/cofactor binding compared with the isolated C-terminal MTase domain. (kaldis2024molecularbasisof pages 11-12)
HEN1 methylation is a defining post-dicing step in plants that stabilizes miRNAs/siRNAs and preserves silencing competence. In the absence of HEN1 activity (hen1 mutants), small RNAs exhibit reduced abundance and 3′ end heterogeneity, including 3′ truncation and uridine-rich tailing. (ji2012regulationofsmall pages 1-3, ding2023micrornaproductionin pages 6-7)
A central consequence of losing HEN1-installed methylation is enhanced susceptibility of small RNAs to 3′-end remodeling:
Genetic evidence ties these reactions to the methylation state: heso1 mutations can partially suppress hen1 phenotypes and reduce uridylation, and hen1 heso1 urt1 backgrounds can abolish U-tailed miRNAs, indicating that HESO1/URT1 are major uridylases acting when HEN1 protection is absent. (ding2023micrornaproductionin pages 7-8, louis2024determinationofgenesa pages 25-28)
Recent syntheses emphasize that methylation is integrated with AGO biology: a subset of miRNAs is loaded into AGO1 in the nucleus and exported as AGO1–miRNA complexes (via CRM1/EXPO1), while other miRNAs appear in the cytoplasm unbound, consistent with multiple export routes (e.g., export of methylated duplexes). This situates HEN1’s action in a broader, spatially organized small-RNA maturation-to-effector continuum. (bajczyk2023recentinsightsinto pages 13-14, ding2023micrornaproductionin pages 1-2)
Direct interaction mapping and mechanistic models place HEN1 in the nuclear miRNA-processing context, associated with the DCL1/HYL1 machinery that resides in nuclear dicing bodies (D-bodies). (baranauske2015functionalmappingof pages 1-2, baranauske2015functionalmappingof pages 9-10)
A functional mapping study using GST pull-downs, yeast two-hybrid, and complex-formation assays demonstrates that HEN1 physically interacts with:
In the same mapping study, no direct interaction between HEN1 and SERRATE was detectable, supporting a model in which HEN1 associates with a DCL1/HYL1-containing assembly while excluding SE at the methylation stage. (baranauske2015functionalmappingof pages 1-2, baranauske2015functionalmappingof pages 8-9)
A 2023 review consolidates a now-standard turnover framework in which HEN1 methylation stabilizes mature miRNAs, while downstream decay involves SDN trimming and HESO1/URT1 uridylation, acting on both AGO-bound and free miRNAs in the cytoplasm. (ding2023micrornaproductionin pages 1-2)
Another 2023 review emphasizes that AGO1 does not simply bind miRNAs but interacts with turnover factors (including uridylation-related enzymes), and highlights multiple routes and constraints for AGO1 loading and miRNA export—context that refines where HEN1’s protective mark matters most. (bajczyk2023recentinsightsinto pages 13-14)
A 2024 review focused on plant RNA tailing frames HEN1 methylation as the upstream “gatekeeper” modification that prevents uridylation/trimming; it also highlights the distinct substrate biases of HESO1 vs URT1, and documents cases where uridylation can alter miRNA functional properties (e.g., reduced repression or slicing activity) and even generate 22-nt forms linked to amplified silencing outputs. (chen2024functionsandmechanisms pages 2-3, chen2024functionsandmechanisms pages 3-5)
A 2024 synthesis of small RNA biogenesis reiterates HEN1’s five-domain architecture and structural model for duplex measurement/recognition, and explicitly connects methylation loss to HESO1-mediated uridylation and SDN-linked destabilization. (li2024thebiosynthesisprocess pages 7-8)
Although the retrieved 2023–2024 sources are largely mechanistic/review-oriented, they collectively justify an applied principle: small RNA stability and silencing persistence in plants can be tuned by manipulating the HEN1 methylation state and/or downstream tailing enzymes (HESO1/URT1) and trimming exonucleases (SDNs), because these factors directly control whether small RNAs are protected or routed into decay. (chen2024functionsandmechanisms pages 2-3, ding2023micrornaproductionin pages 1-2, bajczyk2023recentinsightsinto pages 13-14)
Virus–host interactions commonly target small RNA pathways. A plant-focused review notes that loss of methylation predisposes to uridylation-dependent destabilization and that genetic suppression by heso1 supports the functional importance of tailing in this context; this underpins why viral suppression strategies that impair HEN1 can reprogram small-RNA-mediated defense outputs. (ding2023micrornaproductionin pages 7-8, chen2024functionsandmechanisms pages 2-3)
Across authoritative sources, a consistent interpretation emerges:
Structural evidence supports the expert consensus that HEN1 is specialized for A-form duplex substrates with 2-nt 3′ overhangs and uses modular RNA-binding domains to “measure” the duplex and position a terminal ribose for catalysis—an architecture that explains plant HEN1’s ability to methylate miRNA/siRNA duplexes broadly and non-sequence-specifically. (huang2009structuralinsightsinto pages 1-2, huang2009structuralinsightsinto pages 2-4, huang2009structuralinsightsinto media 62c0a606)
Arabidopsis HEN1 (Q9C5Q8; At4g20910) is a multidomain, SAM-dependent small RNA 3′ 2′-O-methyltransferase that methylates both strands of miRNA/miRNA and siRNA/siRNA duplexes. The modification is a central stabilizing mark that prevents downstream uridylation (HESO1/URT1) and limits trimming (SDNs), thereby protecting small RNAs and ensuring robust RNA silencing. Structural and interaction studies place HEN1 in the nuclear miRNA-processing context (DCL1/HYL1-associated), and 2023–2024 syntheses refine its role within an integrated, compartmentalized miRNA maturation–loading–turnover network and highlight practical levers for tuning small RNA stability and function. (huang2009structuralinsightsinto pages 1-2, baranauske2015functionalmappingof pages 1-2, ding2023micrornaproductionin pages 1-2, chen2024functionsandmechanisms pages 2-3)
| Topic | Summary | Key evidence/citations |
|---|---|---|
| Identity/domains | • Verified target: Arabidopsis thaliana HEN1 / HUA ENHANCER 1 = At4g20910, a plant small-RNA 3'-terminal 2'-O-methyltransferase. • Multidomain architecture: dsRBD1, dsRBD2, La-motif-containing domain (LCD), PPIase-like/FKBP-like domain, C-terminal MTase domain. • Distinct from metazoan HENMT1 and bacterial Hen1 homologs, which differ in size/domain architecture and substrate class. |
(ji2012regulationofsmall pages 1-3, huang2009structuralinsightsinto pages 1-2, peng2018identificationofsubstrates pages 1-2, baranauske2015functionalmappingof pages 1-2) |
| Enzymatic reaction | • Catalyzes transfer of a methyl group from SAM/AdoMet to the 2'-OH of the 3'-terminal ribose of small RNAs; AdoHcy is the product. • In plants, HEN1 methylates both strands of miRNA/miRNA or siRNA/siRNA duplexes in succession. • Reaction is described as metal-dependent in structural/biochemical studies. |
(ji2012regulationofsmall pages 3-4, huang2009structuralinsightsinto pages 1-2, huang2009structuralinsightsinto pages 2-4, baranauske2015functionalmappingof pages 1-2) |
| Substrate specificity | • Preferred substrates are 21–24 nt A-form small-RNA duplexes with 2-nt 3' overhangs. • Recognition is largely non-sequence-specific; HEN1 reads RNA backbone geometry and terminal ribose chemistry rather than bases. • Plant HEN1 acts on miRNA/miRNA and siRNA/siRNA duplexes, unlike animal HEN1/HENMT1, which mainly methylate piRNAs** or AGO2-associated small RNAs. |
(ji2012regulationofsmall pages 3-4, huang2009structuralinsightsinto pages 1-2, huang2009structuralinsightsinto pages 2-4, peng2018identificationofsubstrates pages 1-2) |
| Mechanism/structure determinants | • Crystal structure shows HEN1 binds RNA as a monomer and uses multiple RNA-binding modules to measure duplex length and position one 3' end into the catalytic pocket. • dsRBD1 has a major role in substrate recognition; the LCD/La-motif recognizes the opposite duplex terminus/2-nt overhang; MTase active site engages the methylated strand terminus. • Active-site residues and a coordinated divalent metal support catalysis; plant-specific residues also contribute to 5'-phosphate recognition. |
(huang2009structuralinsightsinto pages 1-2, huang2009structuralinsightsinto pages 2-4, huang2009structuralinsightsinto media 62c0a606) |
| Biological role in pathways | • HEN1 acts in miRNA and siRNA biogenesis, stabilizing nascent duplexes before/around AGO loading. • 2'-O-methylation protects small RNAs from 3' uridylation and 3'→5' degradation, thereby preserving silencing competence. • It is functionally linked to miRNA maturation, RISC assembly, and pathways requiring stable plant small RNAs, including 24-nt siRNA-associated silencing contexts. |
(ji2012regulationofsmall pages 1-3, huang2009structuralinsightsinto pages 1-2, baranauske2015functionalmappingof pages 1-2, ding2023micrornaproductionin pages 1-2) |
| Localization & interactions | • Best-supported context is nuclear miRNA-processing/dicing bodies (D-bodies), where HEN1 joins the DCL1/HYL1 processing assembly after duplex excision. • Direct physical interactions shown with HYL1 and DCL1 by GST pull-down and yeast two-hybrid; no direct interaction detected with SERRATE in the cited study. • No strong direct HEN1–AGO1 interaction is established in the provided evidence, though methylation is tightly coupled to AGO loading/stability pathways. |
(baranauske2015functionalmappingof pages 9-10, baranauske2015functionalmappingof pages 1-2, baranauske2015functionalmappingof pages 8-9, baranauske2015functionalmappingof pages 6-8) |
| Loss-of-function consequences | • hen1 mutants show reduced miRNA abundance, size heterogeneity, 3' truncation, and U-rich tailing/uridylation of small RNAs. • Developmental phenotypes include pleiotropic growth defects such as altered organ size/shape, late flowering, reduced fertility, and light hypersensitivity. • Genetic suppression data show that blocking uridylation pathways (especially HESO1, sometimes URT1) partially rescues hen1 defects. |
(ji2012regulationofsmall pages 1-3, ding2023micrornaproductionin pages 6-7, louis2024determinationofgenesa pages 21-25, louis2024determinationofgenesa pages 25-28, chen2024functionsandmechanisms pages 2-3) |
| Recent 2023-2024 updates | • Recent reviews emphasize that HEN1 methylation is integrated with nuclear AGO1 loading, nucleo-cytoplasmic trafficking, and downstream turnover pathways. • 2024 work/refined models highlight additional turnover factors for unmethylated small RNAs, including ATRM2, and expanded roles for HESO1/URT1/NTPs in tailing, repair, and secondary siRNA regulation. • 2024–2025 modification studies/methods underscore persistent interest in plant small-RNA chemical marks and their dynamics. |
(ding2023micrornaproductionin pages 1-2, louis2024determinationofgenesa pages 21-25, louis2024determinationofgenes pages 21-25, chen2024functionsandmechanisms pages 2-3, chen2024functionsandmechanisms pages 3-5) |
| Applications/biotech relevance | • HEN1 is a key engineering lever for small-RNA stability in plants because methylation status determines susceptibility to uridylation and decay. • It is also a virus-host interface: potyviral HC-Pro can inhibit HEN1, helping explain suppression of antiviral/post-transcriptional silencing. • Knowledge of HEN1 informs crop or synthetic-biology strategies aimed at tuning miRNA/siRNA persistence and silencing output. |
(chen2024functionsandmechanisms pages 2-3, chen2024functionsandmechanisms pages 3-5, ding2023micrornaproductionin pages 7-8) |
| Key quantitative data | • Full-length plant HEN1 kinetics reported as approximately KRNA ≈ 0.22 mM, KSAM ≈ 1.7 mM, kcat ≈ 3.0 min⁻¹ in one mechanistic analysis. • Nature structure solved at 3.1 Å with a 22-nt RNA duplex bound to AdoHcy. • Structural model includes a metal–oxygen distance of about 2.2 Å in active-site coordination in mechanistic modeling. |
(kaldis2024molecularbasisof pages 11-12, huang2009structuralinsightsinto pages 1-2) |
Table: This table condenses the main facts about Arabidopsis thaliana HEN1, including verified identity, catalytic function, pathway role, localization, recent updates, and quantitative evidence. It is designed as a quick-reference summary anchored to the cited context IDs.
References
(huang2009structuralinsightsinto pages 1-2): Ying Huang, Lijuan Ji, Qichen Huang, Dmitry G. Vassylyev, Xuemei Chen, and Jin-Biao Ma. Structural insights into mechanisms of the small rna methyltransferase hen1. Nature, 461:823-827, Oct 2009. URL: https://doi.org/10.1038/nature08433, doi:10.1038/nature08433. This article has 184 citations and is from a highest quality peer-reviewed journal.
(huang2009structuralinsightsinto pages 2-4): Ying Huang, Lijuan Ji, Qichen Huang, Dmitry G. Vassylyev, Xuemei Chen, and Jin-Biao Ma. Structural insights into mechanisms of the small rna methyltransferase hen1. Nature, 461:823-827, Oct 2009. URL: https://doi.org/10.1038/nature08433, doi:10.1038/nature08433. This article has 184 citations and is from a highest quality peer-reviewed journal.
(ji2012regulationofsmall pages 1-3): Lijuan Ji and Xuemei Chen. Regulation of small rna stability: methylation and beyond. Cell Research, 22:624-636, Mar 2012. URL: https://doi.org/10.1038/cr.2012.36, doi:10.1038/cr.2012.36. This article has 328 citations and is from a domain leading peer-reviewed journal.
(peng2018identificationofsubstrates pages 1-2): Ling Peng, Fengjuan Zhang, Renfu Shang, Xueyan Wang, Jiayi Chen, James J. Chou, Jinbiao Ma, Ligang Wu, and Ying Huang. Identification of substrates of the small rna methyltransferase hen1 in mouse spermatogonial stem cells and analysis of its methyl-transfer domain. Journal of Biological Chemistry, 293:9981-9994, Jun 2018. URL: https://doi.org/10.1074/jbc.ra117.000837, doi:10.1074/jbc.ra117.000837. This article has 21 citations and is from a domain leading peer-reviewed journal.
(ding2023micrornaproductionin pages 1-2): Ning Ding and Bailong Zhang. Microrna production in arabidopsis. Frontiers in Plant Science, Jan 2023. URL: https://doi.org/10.3389/fpls.2023.1096772, doi:10.3389/fpls.2023.1096772. This article has 29 citations.
(baranauske2015functionalmappingof pages 1-2): Simona Baranauskė, Milda Mickutė, Alexandra Plotnikova, Andreas Finke, Česlovas Venclovas, Saulius Klimašauskas, and Giedrius Vilkaitis. Functional mapping of the plant small rna methyltransferase: hen1 physically interacts with hyl1 and dicer-like 1 proteins. Nucleic Acids Research, 43:2802-2812, Feb 2015. URL: https://doi.org/10.1093/nar/gkv102, doi:10.1093/nar/gkv102. This article has 103 citations and is from a highest quality peer-reviewed journal.
(ji2012regulationofsmall pages 3-4): Lijuan Ji and Xuemei Chen. Regulation of small rna stability: methylation and beyond. Cell Research, 22:624-636, Mar 2012. URL: https://doi.org/10.1038/cr.2012.36, doi:10.1038/cr.2012.36. This article has 328 citations and is from a domain leading peer-reviewed journal.
(kaldis2024molecularbasisof pages 11-12): Philipp Kaldis and Li Na Zhao. Molecular basis of the reaction mechanism of the methyltransferase henmt1. PLOS ONE, 19:e0293243, Jan 2024. URL: https://doi.org/10.1371/journal.pone.0293243, doi:10.1371/journal.pone.0293243. This article has 2 citations and is from a peer-reviewed journal.
(huang2009structuralinsightsinto media 62c0a606): Ying Huang, Lijuan Ji, Qichen Huang, Dmitry G. Vassylyev, Xuemei Chen, and Jin-Biao Ma. Structural insights into mechanisms of the small rna methyltransferase hen1. Nature, 461:823-827, Oct 2009. URL: https://doi.org/10.1038/nature08433, doi:10.1038/nature08433. This article has 184 citations and is from a highest quality peer-reviewed journal.
(huang2009structuralinsightsinto media 867c9cf3): Ying Huang, Lijuan Ji, Qichen Huang, Dmitry G. Vassylyev, Xuemei Chen, and Jin-Biao Ma. Structural insights into mechanisms of the small rna methyltransferase hen1. Nature, 461:823-827, Oct 2009. URL: https://doi.org/10.1038/nature08433, doi:10.1038/nature08433. This article has 184 citations and is from a highest quality peer-reviewed journal.
(ding2023micrornaproductionin pages 6-7): Ning Ding and Bailong Zhang. Microrna production in arabidopsis. Frontiers in Plant Science, Jan 2023. URL: https://doi.org/10.3389/fpls.2023.1096772, doi:10.3389/fpls.2023.1096772. This article has 29 citations.
(chen2024functionsandmechanisms pages 2-3): Jiwei Chen, Xiaozhen Li, Xianxin Dong, and Xiaoyan Wang. Functions and mechanisms of rna tailing by nucleotidyl transferase proteins in plants. Frontiers in Plant Science, Oct 2024. URL: https://doi.org/10.3389/fpls.2024.1452347, doi:10.3389/fpls.2024.1452347. This article has 3 citations.
(louis2024determinationofgenesa pages 25-28): F Louis. Determination of genes involved in the microrna turnover in arabidopsis thaliana. Unknown journal, 2024.
(ding2023micrornaproductionin pages 7-8): Ning Ding and Bailong Zhang. Microrna production in arabidopsis. Frontiers in Plant Science, Jan 2023. URL: https://doi.org/10.3389/fpls.2023.1096772, doi:10.3389/fpls.2023.1096772. This article has 29 citations.
(bajczyk2023recentinsightsinto pages 13-14): Mateusz Bajczyk, Artur Jarmolowski, Monika Jozwiak, Andrzej Pacak, Halina Pietrykowska, Izabela Sierocka, Aleksandra Swida-Barteczka, Lukasz Szewc, and Zofia Szweykowska-Kulinska. Recent insights into plant mirna biogenesis: multiple layers of mirna level regulation. Plants, 12:342, Jan 2023. URL: https://doi.org/10.3390/plants12020342, doi:10.3390/plants12020342. This article has 79 citations.
(baranauske2015functionalmappingof pages 9-10): Simona Baranauskė, Milda Mickutė, Alexandra Plotnikova, Andreas Finke, Česlovas Venclovas, Saulius Klimašauskas, and Giedrius Vilkaitis. Functional mapping of the plant small rna methyltransferase: hen1 physically interacts with hyl1 and dicer-like 1 proteins. Nucleic Acids Research, 43:2802-2812, Feb 2015. URL: https://doi.org/10.1093/nar/gkv102, doi:10.1093/nar/gkv102. This article has 103 citations and is from a highest quality peer-reviewed journal.
(baranauske2015functionalmappingof pages 6-8): Simona Baranauskė, Milda Mickutė, Alexandra Plotnikova, Andreas Finke, Česlovas Venclovas, Saulius Klimašauskas, and Giedrius Vilkaitis. Functional mapping of the plant small rna methyltransferase: hen1 physically interacts with hyl1 and dicer-like 1 proteins. Nucleic Acids Research, 43:2802-2812, Feb 2015. URL: https://doi.org/10.1093/nar/gkv102, doi:10.1093/nar/gkv102. This article has 103 citations and is from a highest quality peer-reviewed journal.
(baranauske2015functionalmappingof pages 8-9): Simona Baranauskė, Milda Mickutė, Alexandra Plotnikova, Andreas Finke, Česlovas Venclovas, Saulius Klimašauskas, and Giedrius Vilkaitis. Functional mapping of the plant small rna methyltransferase: hen1 physically interacts with hyl1 and dicer-like 1 proteins. Nucleic Acids Research, 43:2802-2812, Feb 2015. URL: https://doi.org/10.1093/nar/gkv102, doi:10.1093/nar/gkv102. This article has 103 citations and is from a highest quality peer-reviewed journal.
(chen2024functionsandmechanisms pages 3-5): Jiwei Chen, Xiaozhen Li, Xianxin Dong, and Xiaoyan Wang. Functions and mechanisms of rna tailing by nucleotidyl transferase proteins in plants. Frontiers in Plant Science, Oct 2024. URL: https://doi.org/10.3389/fpls.2024.1452347, doi:10.3389/fpls.2024.1452347. This article has 3 citations.
(li2024thebiosynthesisprocess pages 7-8): Quan Li, Yanan Wang, Zhihui Sun, Haiyang Li, and Huan Liu. The biosynthesis process of small rna and its pivotal roles in plant development. International Journal of Molecular Sciences, 25:7680, Jul 2024. URL: https://doi.org/10.3390/ijms25147680, doi:10.3390/ijms25147680. This article has 21 citations.
(louis2024determinationofgenesa pages 21-25): F Louis. Determination of genes involved in the microrna turnover in arabidopsis thaliana. Unknown journal, 2024.
(louis2024determinationofgenes pages 21-25): F Louis. Determination of genes involved in the microrna turnover in arabidopsis thaliana. Unknown journal, 2024.
id: Q9C5Q8
gene_symbol: HEN1
product_type: PROTEIN
status: COMPLETE
taxon:
id: NCBITaxon:3702
label: Arabidopsis thaliana
description: HEN1 is the Arabidopsis SAM-dependent small RNA 3' terminal 2'-O-methyltransferase. It methylates
the 2'-OH of the 3'-terminal ribose on miRNA/miRNA-star and siRNA/siRNA-star duplexes after Dicer-like
processing, protecting small RNAs from uridylation, trimming, and degradation. HEN1 acts in the DCL1/HYL1-associated
small-RNA maturation context and supports miRNA, siRNA, ta-siRNA, and antiviral silencing outputs; floral
and leaf patterning phenotypes are downstream consequences of destabilized small RNAs.
existing_annotations:
- term:
id: GO:0034587
label: piRNA processing
evidence_type: IBA
original_reference_id: GO_REF:0000033
review:
summary: HEN1-family proteins include animal piRNA methyltransferases, but Arabidopsis HEN1 methylates
plant miRNA and siRNA duplexes.
action: REMOVE
reason: piRNA processing is an over-transfer from metazoan HEN1/HENMT1 family context and is not supported
for Arabidopsis HEN1.
- term:
id: GO:0008171
label: O-methyltransferase activity
evidence_type: IBA
original_reference_id: GO_REF:0000033
review:
summary: HEN1 is an RNA methyltransferase, but O-methyltransferase activity is less specific than
the small-RNA 2-prime-O-ribose methyltransferase term.
action: MODIFY
reason: Use the small-RNA terminal 2-prime-O-methyltransferase term to capture HEN1 substrate and
reaction specificity.
proposed_replacement_terms:
- &id001
id: GO:0090486
label: small RNA 2'-O-ribose methyltransferase activity
- term:
id: GO:0003755
label: peptidyl-prolyl cis-trans isomerase activity
evidence_type: IEA
original_reference_id: GO_REF:0000002
review:
summary: HEN1 includes a PPIase-like/FKBP-like domain, but its curated function is small-RNA methyltransferase
activity.
action: REMOVE
reason: The PPIase-like/FKBP-like domain is present, but no evidence supports HEN1 peptidyl-prolyl
isomerase catalytic activity; the supported biochemical function is small-RNA methyltransferase
activity.
- term:
id: GO:0005634
label: nucleus
evidence_type: IEA
original_reference_id: GO_REF:0000044
review:
summary: HEN1 is associated with the nuclear DCL1/HYL1 small-RNA maturation context.
action: ACCEPT
reason: The best-supported site for HEN1 action is nuclear small-RNA processing/methylation before
or around AGO loading.
supported_by:
- reference_id: file:ARATH/HEN1/HEN1-deep-research-falcon.md
supporting_text: Direct interaction mapping and mechanistic models place HEN1 in the nuclear miRNA-processing
context.
- term:
id: GO:0008171
label: O-methyltransferase activity
evidence_type: IEA
original_reference_id: GO_REF:0000002
review:
summary: HEN1 is an RNA methyltransferase, but O-methyltransferase activity is less specific than
the small-RNA 2-prime-O-ribose methyltransferase term.
action: MODIFY
reason: Use the small-RNA terminal 2-prime-O-methyltransferase term to capture HEN1 substrate and
reaction specificity.
proposed_replacement_terms:
- *id001
- term:
id: GO:0008173
label: RNA methyltransferase activity
evidence_type: IEA
original_reference_id: GO_REF:0000002
review:
summary: HEN1 is an RNA methyltransferase, but RNA methyltransferase activity is less specific than
the small-RNA 2-prime-O-ribose methyltransferase term.
action: MODIFY
reason: Use the small-RNA terminal 2-prime-O-methyltransferase term to capture HEN1 substrate and
reaction specificity.
proposed_replacement_terms:
- *id001
- term:
id: GO:0016070
label: RNA metabolic process
evidence_type: IEA
original_reference_id: GO_REF:0000117
review:
summary: HEN1 participates in small-RNA metabolism by methylating mature miRNA/siRNA duplexes.
action: MODIFY
reason: RNA metabolic process is too broad; small-RNA methylation and miRNA/siRNA processing-stabilization
outputs are more informative.
proposed_replacement_terms:
- *id001
- id: GO:0035196
label: miRNA processing
- term:
id: GO:0090486
label: small RNA 2'-O-ribose methyltransferase activity
evidence_type: IEA
original_reference_id: GO_REF:0000003
review:
summary: HEN1 catalyzes terminal 2-prime-O-ribose methylation of plant small RNAs. The HEN1
crystal structure (PDB 3HTX) shows the SAH cofactor product bound in the Rossmann MTase domain
as in classical SAM-dependent methyltransferases, with a catalytic Mg ion coordinating the
3-prime-terminal 2-prime and 3-prime hydroxyls, consistent with 2-prime-O-ribose methyl transfer.
action: ACCEPT
reason: This is the most specific molecular-function term for Arabidopsis HEN1.
supported_by:
- reference_id: file:ARATH/HEN1/HEN1-deep-research-falcon.md
supporting_text: HEN1 is a small RNA 3-prime-terminal 2-prime-O-methyltransferase.
- reference_id: file:ARATH/HEN1/HEN1-uniprot.txt
supporting_text: Reaction=small RNA 3'-end nucleotide + S-adenosyl-L-methionine = small RNA 3'-end
2'-O-methylnucleotide + S-adenosyl-L-homocysteine + H(+);
- reference_id: file:interpro/panther/PTHR21404/PTHR21404-entries.csv
supporting_text: Q9C5Q8,Small RNA 2'-O-methyltransferase
- reference_id: PMID:19812675
supporting_text: The MTase domain of HEN1 adopts a core α/β Rossmann structure, in which the
cofactor product AdoHcy is bound as in classical S-adenosyl-l-methionine (AdoMet)-dependent
MTases
- reference_id: PMID:19812675
supporting_text: both the 2′ and 3′ hydroxyls of G22m and the side chains of four invariant
residues (E796, E799, H800 and H860) are coordinated to a metal ion, Mg2+
- term:
id: GO:0090486
label: small RNA 2'-O-ribose methyltransferase activity
evidence_type: IEA
original_reference_id: GO_REF:0000116
review:
summary: HEN1 catalyzes terminal 2-prime-O-ribose methylation of plant small RNAs. The 3.1 A
crystal structure of full-length Arabidopsis HEN1 (PDB 3HTX) bound to a 22-nucleotide small
RNA duplex with the cofactor product SAH (S-adenosyl-L-homocysteine) and Mg directly visualizes
the substrate as a small-RNA duplex and confirms terminal 2-prime-O-methyltransferase chemistry.
action: ACCEPT
reason: This is the most specific molecular-function term for Arabidopsis HEN1.
supported_by:
- reference_id: file:ARATH/HEN1/HEN1-deep-research-falcon.md
supporting_text: HEN1 is a small RNA 3-prime-terminal 2-prime-O-methyltransferase.
- reference_id: file:ARATH/HEN1/HEN1-uniprot.txt
supporting_text: Reaction=small RNA 3'-end nucleotide + S-adenosyl-L-methionine = small RNA 3'-end
2'-O-methylnucleotide + S-adenosyl-L-homocysteine + H(+);
- reference_id: file:interpro/panther/PTHR21404/PTHR21404-entries.csv
supporting_text: Q9C5Q8,Small RNA 2'-O-methyltransferase
- reference_id: PMID:19812675
supporting_text: report the 3.1 A crystal structure of full-length HEN1 from Arabidopsis in
complex with a 22-nucleotide small RNA duplex and cofactor product S-adenosyl-l-homocysteine
- reference_id: PMID:19812675
supporting_text: A subset of small RNAs, such as microRNAs and small interfering RNAs
(siRNAs) in plants, Piwi-interacting RNAs in animals and siRNAs in Drosophila, requires
an additional crucial step for their maturation; that is, 2'-O-methylation on the 3' terminal
nucleotide. A conserved S-adenosyl-l-methionine-dependent RNA methyltransferase, HUA ENHANCER
1 (HEN1), and its homologues are responsible for this specific modification.
- term:
id: GO:0010267
label: ta-siRNA processing
evidence_type: IMP
original_reference_id: PMID:15469823
review:
summary: HEN1 supports ta-siRNA processing by methylating and stabilizing the small-RNA duplexes that
feed these pathways.
action: KEEP_AS_NON_CORE
reason: HEN1 is required for stable miRNA/siRNA pathway outputs, but the direct catalytic step is
small-RNA 2-prime-O-methylation rather than Dicer cleavage or AGO-mediated target repression.
supported_by:
- reference_id: file:ARATH/HEN1/HEN1-uniprot.txt
supporting_text: Can methylate 3'-end of microRNAs (miRNAs), small interfering RNAs (siRNas) and
trans-acting small interfering RNAs (ta-siRNAs).
- term:
id: GO:0005737
label: cytoplasm
evidence_type: ISM
original_reference_id: GO_REF:0000122
review:
summary: The AtSubP-inferred cytoplasm row lacks positive experimental support for HEN1 cytoplasmic
localization.
action: UNDECIDED
reason: HEN1's best-supported context is small-RNA methylation around nuclear miRNA/siRNA maturation;
downstream cytoplasmic small-RNA turnover is not sufficient evidence for a cytoplasm localization
annotation.
- term:
id: GO:0010305
label: leaf vascular tissue pattern formation
evidence_type: IMP
original_reference_id: PMID:22623415
review:
summary: hen1 mutants affect leaf vascular tissue pattern formation.
action: KEEP_AS_NON_CORE
reason: The developmental process is a downstream phenotype of altered miRNA/siRNA stability rather
than a direct HEN1 function.
- term:
id: GO:0010589
label: leaf proximal/distal pattern formation
evidence_type: IMP
original_reference_id: PMID:22623415
review:
summary: hen1 mutants affect leaf proximal/distal pattern formation.
action: KEEP_AS_NON_CORE
reason: The developmental process is a downstream phenotype of altered miRNA/siRNA stability rather
than a direct HEN1 function.
- term:
id: GO:0009616
label: RNAi-mediated antiviral immune response
evidence_type: IMP
original_reference_id: PMID:17090584
review:
summary: HEN1 contributes to virus-induced silencing and viral-small-RNA stability through terminal
small-RNA methylation.
action: KEEP_AS_NON_CORE
reason: The antiviral role is a pathway output of HEN1-mediated small-RNA stabilization; the direct
catalytic function remains small-RNA 2-prime-O-methylation rather than Dicing or Argonaute effector
activity.
supported_by:
- reference_id: PMID:17090584
supporting_text: DCL4 in conjunction with RDR6 and HEN1 specifically facilitates extensive virus-induced
silencing in new growth.
- term:
id: GO:0005634
label: nucleus
evidence_type: IDA
original_reference_id: PMID:17442570
review:
summary: The cited D-body paper places plant miRNA processing in the nucleus but directly images
DCL1/HYL1/SE rather than HEN1.
action: ACCEPT
reason: Although PMID:17442570 directly images other D-body proteins, HEN1 has independent support
for the same nuclear miRNA-processing context, so the nucleus annotation is retained consistently.
supported_by:
- reference_id: file:ARATH/HEN1/HEN1-deep-research-falcon.md
supporting_text: Direct interaction mapping and mechanistic models place HEN1 in the nuclear miRNA-processing
context.
- term:
id: GO:0005737
label: cytoplasm
evidence_type: IDA
original_reference_id: PMID:17442570
review:
summary: The cited D-body paper contrasts animal cytoplasmic miRNA processing with plant nuclear
processing and does not directly support HEN1 cytoplasmic localization.
action: UNDECIDED
reason: HEN1 may influence cytoplasmic small-RNA turnover indirectly, but PMID:17442570 does not provide
direct HEN1 cytoplasm evidence.
- term:
id: GO:0008173
label: RNA methyltransferase activity
evidence_type: IDA
original_reference_id: PMID:15705854
review:
summary: Experimental Arabidopsis work supports HEN1-dependent methylation/protection of miRNAs and
siRNAs. The HEN1 crystal structure (PDB 3HTX) confirms it is a SAM-dependent RNA methyltransferase,
with the AdoHcy (SAH) product bound in a Rossmann MTase domain as in classical AdoMet-dependent
MTases.
action: MODIFY
reason: Use the small-RNA terminal 2-prime-O-methyltransferase term to capture HEN1 substrate and
reaction specificity.
proposed_replacement_terms:
- *id001
supported_by:
- reference_id: file:ARATH/HEN1/HEN1-deep-research-falcon.md
supporting_text: HEN1 catalyzes transfer of a methyl group from AdoMet to the 2-prime-OH of the
3-prime-terminal nucleotide of small RNAs.
- reference_id: file:ARATH/HEN1/HEN1-uniprot.txt
supporting_text: Methyltransferase that adds a methyl group to the ribose of the last nucleotide
of small RNAs (sRNAs).
- reference_id: PMID:19812675
supporting_text: A conserved S-adenosyl-l-methionine-dependent RNA methyltransferase, HUA ENHANCER
1 (HEN1), and its homologues are responsible for this specific modification.
- reference_id: PMID:19812675
supporting_text: The MTase domain of HEN1 adopts a core α/β Rossmann structure, in which the
cofactor product AdoHcy is bound as in classical S-adenosyl-l-methionine (AdoMet)-dependent
MTases
- term:
id: GO:0008173
label: RNA methyltransferase activity
evidence_type: IMP
original_reference_id: PMID:16111943
review:
summary: Experimental Arabidopsis work supports HEN1-dependent methylation/protection of miRNAs and
siRNAs. The HEN1 crystal structure (PDB 3HTX) shows a catalytic Mg2+ ion coordinated by the 2-prime
and 3-prime hydroxyls of the 3-prime-terminal nucleotide and four invariant active-site residues,
defining a Mg2+-dependent 2-prime-O-methylation mechanism.
action: MODIFY
reason: Use the small-RNA terminal 2-prime-O-methyltransferase term to capture HEN1 substrate and
reaction specificity.
proposed_replacement_terms:
- *id001
supported_by:
- reference_id: file:ARATH/HEN1/HEN1-deep-research-falcon.md
supporting_text: HEN1 catalyzes transfer of a methyl group from AdoMet to the 2-prime-OH of the
3-prime-terminal nucleotide of small RNAs.
- reference_id: file:ARATH/HEN1/HEN1-uniprot.txt
supporting_text: Methyltransferase that adds a methyl group to the ribose of the last nucleotide
of small RNAs (sRNAs).
- reference_id: PMID:19812675
supporting_text: Metal ion coordination by both 2' and 3' hydroxyls on the 3'-terminal nucleotide
and four invariant residues in the active site of the methyltransferase domain suggests a novel
Mg(2+)-dependent 2'-O-methylation mechanism.
- reference_id: PMID:19812675
supporting_text: both the 2′ and 3′ hydroxyls of G22m and the side chains of four invariant
residues (E796, E799, H800 and H860) are coordinated to a metal ion, Mg2+
- term:
id: GO:0035279
label: miRNA-mediated gene silencing by mRNA destabilization
evidence_type: IMP
original_reference_id: PMID:15851028
review:
summary: HEN1 supports miRNA-mediated gene silencing by mRNA destabilization by methylating and stabilizing
the small-RNA duplexes that feed these pathways.
action: KEEP_AS_NON_CORE
reason: HEN1 is required for stable miRNA/siRNA pathway outputs, but the direct catalytic step is
small-RNA 2-prime-O-methylation rather than Dicer cleavage or AGO-mediated target repression.
supported_by:
- reference_id: file:ARATH/HEN1/HEN1-uniprot.txt
supporting_text: Can methylate 3'-end of microRNAs (miRNAs), small interfering RNAs (siRNas) and
trans-acting small interfering RNAs (ta-siRNAs).
- term:
id: GO:0035196
label: miRNA processing
evidence_type: IMP
original_reference_id: PMID:12747833
review:
summary: HEN1 supports miRNA processing by methylating and stabilizing the small-RNA duplexes that
feed these pathways.
action: ACCEPT
reason: HEN1 methylation is a required post-dicing maturation/protection step in plant miRNA biogenesis,
so miRNA processing is supported even though the more specific catalytic role is small-RNA
2-prime-O-methylation.
supported_by:
- reference_id: file:ARATH/HEN1/HEN1-uniprot.txt
supporting_text: Can methylate 3'-end of microRNAs (miRNAs), small interfering RNAs (siRNas) and
trans-acting small interfering RNAs (ta-siRNAs).
- reference_id: file:ARATH/HEN1/HEN1-deep-research-falcon.md
supporting_text: HEN1 methylation is a defining post-dicing step in plants that stabilizes miRNAs
and siRNAs.
- reference_id: PMID:19812675
supporting_text: A subset of small RNAs, such as microRNAs and small interfering RNAs
(siRNAs) in plants, Piwi-interacting RNAs in animals and siRNAs in Drosophila, requires
an additional crucial step for their maturation; that is, 2'-O-methylation on the 3' terminal
nucleotide.
- term:
id: GO:0009909
label: regulation of flower development
evidence_type: IMP
original_reference_id: PMID:11917084
review:
summary: hen1 mutants affect regulation of flower development.
action: KEEP_AS_NON_CORE
reason: The developmental process is a downstream phenotype of altered miRNA/siRNA stability rather
than a direct HEN1 function.
- term:
id: GO:0010093
label: specification of floral organ identity
evidence_type: IMP
original_reference_id: PMID:11874905
review:
summary: hen1 mutants affect specification of floral organ identity.
action: KEEP_AS_NON_CORE
reason: The developmental process is a downstream phenotype of altered miRNA/siRNA stability rather
than a direct HEN1 function.
references:
- id: GO_REF:0000002
title: Gene Ontology annotation through association of InterPro records with GO terms
findings: []
- id: GO_REF:0000003
title: Gene Ontology annotation based on Enzyme Commission mapping
findings: []
- id: GO_REF:0000033
title: Annotation inferences using phylogenetic trees
findings: []
- id: GO_REF:0000044
title: Gene Ontology annotation based on UniProtKB/Swiss-Prot Subcellular Location vocabulary mapping,
accompanied by conservative changes to GO terms applied by UniProt
findings: []
- id: GO_REF:0000116
title: Automatic Gene Ontology annotation based on Rhea mapping
findings: []
- id: GO_REF:0000117
title: Electronic Gene Ontology annotations created by ARBA machine learning models
findings: []
- id: GO_REF:0000122
title: AtSubP analysis
findings: []
- id: PMID:11874905
title: HEN1 functions pleiotropically in Arabidopsis development and acts in C function in the flower.
findings: []
- id: PMID:11917084
title: Formation of corymb-like inflorescences due to delay in bolting and flower development in the
corymbosa2 mutant of Arabidopsis.
findings: []
- id: PMID:12747833
title: 'Arabidopsis HEN1: a genetic link between endogenous miRNA controlling development and siRNA
controlling transgene silencing and virus resistance.'
findings:
- statement: This source links HEN1 genetically to endogenous miRNA and siRNA silencing pathways.
- id: PMID:15469823
title: Endogenous trans-acting siRNAs regulate the accumulation of Arabidopsis mRNAs.
findings: []
- id: PMID:15705854
title: Methylation as a crucial step in plant microRNA biogenesis.
findings:
- statement: This source supports HEN1-dependent methylation as a crucial step in plant miRNA biogenesis.
- id: PMID:15851028
title: microRNA-directed phasing during trans-acting siRNA biogenesis in plants.
findings: []
- id: PMID:16111943
title: Methylation protects miRNAs and siRNAs from a 3'-end uridylation activity in Arabidopsis.
findings:
- statement: This source supports HEN1 methylation protecting miRNAs and siRNAs from 3-prime uridylation/degradation.
- id: PMID:17090584
title: Four plant Dicers mediate viral small RNA biogenesis and DNA virus induced silencing.
findings:
- statement: This source supports Dicer-mediated viral small RNA production and a DCL4/RDR6/HEN1 requirement
for extensive virus-induced silencing in new growth; HEN1 involvement should be interpreted as small-RNA
stabilization rather than antiviral Dicer activity.
- id: PMID:17442570
title: Identification of nuclear dicing bodies containing proteins for microRNA biogenesis in living
Arabidopsis plants.
findings:
- statement: This source supports DCL1/HYL1/SE nuclear dicing bodies but does not directly image HEN1
localization.
- id: PMID:19812675
title: Structural insights into mechanisms of the small RNA methyltransferase HEN1.
findings:
- statement: The 3.1 A crystal structure of full-length Arabidopsis HEN1 (PDB 3HTX) in complex with
a 22-nucleotide small RNA duplex, the cofactor product S-adenosyl-L-homocysteine (SAH), and a
catalytic Mg2+ ion reveals a SAM-dependent, Mg2+-dependent 3'-terminal 2'-O-methylation mechanism.
reference_review:
relevance: HIGH
correctness: VERIFIED
review_notes: PubMed-verified structure paper (PDB 3HTX) showing full-length Arabidopsis HEN1 bound
to a small RNA duplex with SAH and Mg2+, directly supporting its SAM-dependent, Mg2+-dependent
small-RNA 3'-terminal 2'-O-methyltransferase activity.
- id: PMID:22623415
title: The microRNA pathway genes AGO1, HEN1 and HYL1 participate in leaf proximal-distal, venation
and stomatal patterning in Arabidopsis.
findings: []
- id: file:ARATH/HEN1/HEN1-deep-research-falcon.md
title: Falcon deep research for Arabidopsis HEN1
findings:
- statement: HEN1 is a SAM-dependent small-RNA 3-prime-terminal 2-prime-O-methyltransferase that protects
miRNAs and siRNAs from uridylation/trimming.
- id: file:ARATH/HEN1/HEN1-uniprot.txt
title: UniProt record for Arabidopsis HEN1
findings:
- statement: UniProt describes HEN1 as methylating the last nucleotide of miRNAs, siRNAs, and ta-siRNAs
to protect small RNAs from uridylation and degradation.
- id: file:interpro/panther/PTHR21404/PTHR21404-entries.csv
title: PANTHER PTHR21404 HEN1 family entries
findings:
- statement: Arabidopsis HEN1 is the single Arabidopsis member in this HEN1/small RNA 2-prime-O-methyltransferase
family context.
core_functions:
- description: Small-RNA terminal 2-prime-O-methyltransferase activity. HEN1 transfers a methyl group
from SAM to the 2-prime-OH of the 3-prime-terminal ribose of miRNA/miRNA-star and siRNA/siRNA-star
duplexes.
molecular_function: *id001
directly_involved_in:
- id: GO:0035196
label: miRNA processing
supported_by:
- reference_id: file:ARATH/HEN1/HEN1-deep-research-falcon.md
supporting_text: HEN1 is a small RNA 3-prime-terminal 2-prime-O-methyltransferase.
- reference_id: file:ARATH/HEN1/HEN1-uniprot.txt
supporting_text: Methyltransferase that adds a methyl group to the ribose of the last nucleotide of
small RNAs (sRNAs).
- reference_id: file:interpro/panther/PTHR21404/PTHR21404-entries.csv
supporting_text: Q9C5Q8,Small RNA 2'-O-methyltransferase
- description: Protection and stabilization of plant small RNAs. HEN1-installed methylation protects miRNAs
and siRNAs from HESO1/URT1-dependent uridylation, trimming, and degradation, preserving RNA-silencing
competence.
molecular_function:
id: GO:0090486
label: small RNA 2'-O-ribose methyltransferase activity
directly_involved_in:
- id: GO:0035196
label: miRNA processing
supported_by:
- reference_id: file:ARATH/HEN1/HEN1-deep-research-falcon.md
supporting_text: HEN1 methylation is a defining post-dicing step in plants that stabilizes miRNAs
and siRNAs.
- reference_id: file:ARATH/HEN1/HEN1-uniprot.txt
supporting_text: This protects the 3'-end of sRNAs from uridylation activity and subsequent degradation.
proposed_new_terms:
- proposed_name: small RNA 3-prime-end protection by 2-prime-O-methylation
proposed_definition: A small-RNA metabolic process in which methylation of the 2-prime-OH
of the 3-prime-terminal ribose protects mature small RNAs from uridylation, trimming,
and exonucleolytic decay.
justification: HEN1's direct molecular function is captured by small RNA 2-prime-O-ribose
methyltransferase activity, but the current core-function summary also needs to distinguish
the downstream protective effect of that modification. A process term for small-RNA
3-prime-end protection would avoid reusing the same catalytic molecular-function term
for both methyl transfer and stabilization.
proposed_parent:
id: GO:0006396
label: RNA processing
suggested_questions:
- question: Should piRNA-processing IBA transfer be excluded systematically for plant HEN1 proteins without
piRNA pathway evidence?
- question: Should GO add a process term such as small RNA 3-prime-end protection by 2-prime-O-methylation
for HEN1-dependent protection from uridylation, trimming, and decay?
suggested_experiments:
- description: Quantify methylation efficiency of Arabidopsis HEN1 on defined miRNA, siRNA, and ta-siRNA
duplex substrates with varying overhang geometry.
- description: Map the compartment and timing of HEN1 methylation relative to DCL1/HYL1 processing and
AGO1 loading using endogenous tagged proteins.