Plant miRNA biogenesis and AGO1 RISC loading

In Arabidopsis thaliana, microRNAs (miRNAs) are transcribed by RNA polymerase II as capped and polyadenylated primary transcripts (pri-miRNAs) that fold into imperfect stem-loop hairpins. Unlike animals, which split processing between nuclear Drosha and cytoplasmic Dicer, plants carry out both cleavage steps in the nucleus with a single RNase III enzyme, DICER-LIKE1 (DCL1). DCL1 works in a microprocessor with the double-stranded RNA-binding protein HYPONASTIC LEAVES1 (HYL1, also called DRB1) and the C2H2 zinc-finger protein SERRATE (SE). The three proteins meet in nuclear dicing bodies (D-bodies) and nucleoplasm. DCL1 alone can cut miRNA precursors, but most of its products are then misplaced. HYL1 and SE raise the rate of DCL1 cleavage and position the cuts, so that the correct 21-nt miRNA/miRNA* duplex is released. The small-RNA methyltransferase HUA ENHANCER1 (HEN1) then adds a 2'-O-methyl group to the 3'-terminal ribose of each strand of the duplex. This methyl mark protects the miRNA from 3' uridylation, trimming and degradation. The guide strand is then loaded into ARGONAUTE1 (AGO1), the main plant miRNA effector. AGO1 cleaves complementary target mRNAs through its PIWI-domain slicer activity and can also repress their translation. The miRNA-AGO1 complex is the output of this module.

MODULE:plant_mirna_biogenesisDRAFTCONCRETEBiological Processmodules/plant_mirna_biogenesis.yaml

miRNA processingGO:0035196 miRNA-mediated post-transcriptional gene silencingGO:0035195

PMID:17442570

Identification of nuclear dicing bodies containing proteins for microRNA biogenesis in living Arabidopsis plants.

Both processing steps occur in the plant nucleus by DCL1 with SE and HYL1; the miRNAs are then methylated by HEN1 and loaded onto AGO1.

in plant cells both steps occur in the nucleus via the enzyme Dicer-like1 (DCL1)

PMID:16144699

MicroRNA biogenesis and function in plants.

Plant miRNA biogenesis adds a HEN1 methylation step to the two RNase III cleavage steps and RISC incorporation shared with animals.

the biogenesis of plant miRNAs consists of an additional step, i.e., the miRNAs are methylated on the ribose of the last nucleotide by the miRNA methyltransferase HEN1.

PMID:18632569

The RNA-binding proteins HYL1 and SE promote accurate in vitro processing of pri-miRNA by DCL1.

Reconstitution with recombinant proteins shows that DCL1 alone cleaves miRNA precursors inaccurately and that HYL1 and SE raise the rate and accuracy of cleavage.

We show that recombinant HYL1 and SE proteins accelerate the rate of DCL1-mediated cleavage of pre- and pri-miR167b substrates and promote accurate processing.

file:genes/ARATH/DCL1/DCL1-ai-review.yaml

Arabidopsis DCL1 gene review

Core functions are ribonuclease III activity (GO:0004525) and double-stranded RNA binding (GO:0003725) in primary miRNA processing within the DCL1-HYL1-SE microprocessor.

file:genes/ARATH/DRB1/DRB1-ai-review.yaml

Arabidopsis DRB1/HYL1 gene review

Core functions are double-stranded RNA binding (GO:0003725) and noncatalytic ribonuclease activator activity (GO:0170054) toward DCL1; the IBA ribonuclease III activity row is removed.

file:genes/ARATH/HEN1/HEN1-ai-review.yaml

Arabidopsis HEN1 gene review

Core function is small RNA 2'-O-ribose methyltransferase activity (GO:0090486) on miRNA/miRNA* and siRNA/siRNA* duplexes.

file:genes/ARATH/AGO1/AGO1-ai-review.yaml

Arabidopsis AGO1 gene review

Core functions are miRNA/siRNA-guided RNA endonuclease (slicer) activity (GO:0004521) in miRNA-mediated post-transcriptional gene silencing, siRNA binding, and translation repressor activity (GO:0030371) in miRNA-mediated inhibition of translation at the ER.

Boundary. The module starts at the pri-miRNA hairpin and ends with the miRNA-loaded AGO1 effector and its two outputs, target slicing and translational repression at the ER. MIR gene transcription by RNA polymerase II and its regulators (for example DAWDLE, CDC5, NOT2, and HYL1's own role at MIR loci) are upstream and are not modelled. Other accessory microprocessor factors (TGH, CPL1, which dephosphorylates HYL1, and the cap-binding complex CBP20/CBP80) and HASTY-dependent export are also not modelled. None of these has an Arabidopsis gene review yet. The specific 3'-to-5' exonucleases (SDN1) and uridylyltransferases (HESO1, URT1) that act on unmethylated or AGO1-bound miRNAs are miRNA turnover and are out of scope. RDR6 is deliberately excluded. RDR6 (UniProtKB:Q9SG02, reviewed in genes/ARATH/RDR6) is an RNA-dependent RNA polymerase that converts AGO-cleaved or aberrant transcripts into dsRNA for DCL4/DCL2-dependent secondary siRNA (tasiRNA/phasiRNA) production with SGS3. It acts downstream of miRNA-guided cleavage (for example miR173-AGO1 at TAS1/TAS2 and miR390-AGO7 at TAS3). It has no role in making miRNAs. It belongs in a separate secondary-siRNA amplification module. That module would take miRISC cleavage of TAS transcripts as its input, and its siRNAs would also be HEN1-methylated and AGO1-loaded. Likewise, DCL1-produced inverted-repeat siRNAs and the heterochromatic DCL3/RDR2/AGO4 pathway are out of scope. The HYL1 ribonuclease activator assertion follows the DRB1 gene review, which removes the IBA ribonuclease III activity row because HYL1 has no RNase III domain. No production GO-CAMs in gocams/index.tsv contain these Arabidopsis proteins. SERRATE (UniProtKB:Q9ZVD0) has no gene review yet. It is the only grounding in this module without one.

4Nodes

3Parts

0Variant Sets

0Variants

8Annotons

2Connections

Derived QC

Recommended-field compliance

100.0% recommended fields populated

All recommended fields populated.

Module deep research

✗ none found

No MODULE:plant_mirna_biogenesis deep-research report alongside the module YAML.

Leaf nodes lacking representative members

✓ every leaf node grounds to a representative protein.

Template conformance

✓ every declared conforms_to bundle matches its template motif.

Function consistency and core coverage

7 gene-function check(s) reflected in core functions · 0 conflict(s) · 1 evidence or coverage gap(s) (8 comparison(s))

Retained noncore functions are reported separately; they do not require promotion to core functions. Support from representative genes does not establish conservation across the whole family. Missing reviews or identifiers leave a claim unverified.

Annoton	Participant	Function	Assessment
dcl1_rnase3	DCL1 UniProtKB:Q9SP32 Gene	ribonuclease III activity GO:0004525	Core Supported The GO molecular function is reflected in core_functions (exact term or a reviewed subclass).
hyl1_dsrna_binding	HYL1 (DRB1) UniProtKB:O04492 Gene	double-stranded RNA binding GO:0003725	Core Supported The GO molecular function is reflected in core_functions (exact term or a reviewed subclass).
hyl1_dcl1_activator	HYL1 (DRB1) UniProtKB:O04492 Gene	ribonuclease activator activity GO:0170054	Core Supported The GO molecular function is reflected in core_functions (exact term or a reviewed subclass).
se_dcl1_activator	SERRATE (SE) UniProtKB:Q9ZVD0 Gene	ribonuclease activator activity GO:0170054	Core Supported The GO molecular function is reflected in core_functions (exact term or a reviewed subclass).
hen1_mtase	HEN1 UniProtKB:Q9C5Q8 Gene	small RNA 2'-O-ribose methyltransferase activity GO:0090486	Core Supported The GO molecular function is reflected in core_functions (exact term or a reviewed subclass).
ago1_guide_binding	AGO1 UniProtKB:O04379 Gene	miRNA binding GO:0035198	Annotation Supported A retained annotation or curated replacement supports this function, but core_functions does not yet reflect it.
ago1_slicer	AGO1 UniProtKB:O04379 Gene	endoribonuclease activity, cleaving miRNA-paired mRNA GO:0090624	Core Supported The GO molecular function is reflected in core_functions (exact term or a reviewed subclass).
ago1_translational_repression	AGO1 UniProtKB:O04379 Gene	translation repressor activity GO:0030371	Core Supported The GO molecular function is reflected in core_functions (exact term or a reviewed subclass).

Gene-review completeness (5/5 grounded genes reviewed)

5 complete review(s) · 4 with deep research · 0 missing review · 1 reviewed but lacking deep research

Gene	Review	Complete	Deep research
AGO1 O04379	✓	✓	✓
DCL1 Q9SP32	✓	✓	✓
DRB1 O04492	✓	✓	✓
HEN1 Q9C5Q8	✓	✓	✓
SERRATE Q9ZVD0	✓	✓	✗

Details

Context

Arabidopsis thalianaNCBITaxon:3702

Plant miRNA biogenesis (DCL1-HYL1-SE processing, HEN1 methylation, AGO1 loading)Biological Processplant_mirna_biogenesis

Concrete Arabidopsis module with three ordered steps: nuclear processing of pri-miRNA hairpins by the DCL1-HYL1-SE microprocessor, HEN1 2'-O-methylation of the miRNA/miRNA* duplex, and loading of the guide strand into AGO1 to form the miRNA-induced silencing complex (miRISC) that slices target mRNAs and represses their translation.

miRNA processingGO:0035196 miRNA-mediated post-transcriptional gene silencingGO:0035195

Context

Arabidopsis thalianaNCBITaxon:3702

Connections

dcl1_microprocessor -> hen1_methylation Provides Input For

The DCL1-HYL1-SE microprocessor releases the miRNA/miRNA* duplex, which is the substrate HEN1 methylates.

PMID:16889646

we provide evidence that SE and HYL1 probably act with DCL1 in processing pri-miRNAs before HEN1 in miRNA biogenesis.

hen1_methylation -> ago1_mirisc Provides Input For

The methylated miRNA guide strand is loaded into AGO1.

PMID:17442570

Plant miRNA/miRNA* duplexes generated by DCL1 are methylated by HEN1 at their 3’ ends

Part 1: nuclear pri-miRNA and pre-miRNA processing (microprocessor)

DCL1-HYL1-SE microprocessor in nuclear dicing bodiesProtein Complexdcl1_microprocessor

DCL1 makes both cuts that release the miRNA/miRNA* duplex from the pri-miRNA stem-loop. HYL1 binds the precursor stem through its tandem N-terminal dsRNA-binding domains and binds DCL1 through the second DCL1 dsRNA-binding domain. SE, a C2H2 zinc-finger protein that also works in pre-mRNA splicing, binds HYL1. HYL1 and SE do not cut RNA themselves. They speed up DCL1 and place its cuts at the right positions. The three proteins interact in nuclear dicing bodies. A DCL1 truncation that lacks the second dsRNA-binding domain (dcl1-9) neither binds HYL1 nor enters D-bodies.

primary miRNA processingGO:0031053 pre-miRNA processingGO:0031054

Context

nuclear dicing bodyGO:0010445

Annotons

DCL1: nuclear RNase III that cuts pri- and pre-miRNA hairpins

dcl1_rnase3

Participant: Gene Product: DCL1

Gene Product:

DCL1UniProtKB:Q9SP32

Function

ribonuclease III activityGO:0004525

Processes

primary miRNA processingGO:0031053 pre-miRNA processingGO:0031054

Locations

nuclear dicing bodyGO:0010445

HYL1/DRB1: dsRNA-binding recognition of the miRNA precursor stem

hyl1_dsrna_binding

Participant: Gene Product: HYL1 (DRB1)

Gene Product:

HYL1 (DRB1)UniProtKB:O04492

Function

double-stranded RNA bindingGO:0003725

Processes

pre-miRNA processingGO:0031054

Locations

nuclear dicing bodyGO:0010445

HYL1/DRB1: noncatalytic activator of DCL1 cleavage rate and accuracy

hyl1_dcl1_activator

Participant: Gene Product: HYL1 (DRB1)

Gene Product:

HYL1 (DRB1)UniProtKB:O04492

Function

ribonuclease activator activityGO:0170054

Targets: DCL1UniProtKB:Q9SP32

Processes

miRNA processingGO:0035196

Locations

nuclear dicing bodyGO:0010445

SE: zinc-finger cofactor that raises DCL1 cleavage rate and accuracy

se_dcl1_activator

Participant: Gene Product: SERRATE (SE)

Gene Product:

SERRATE (SE)UniProtKB:Q9ZVD0

Function

ribonuclease activator activityGO:0170054

Targets: DCL1UniProtKB:Q9SP32

Processes

primary miRNA processingGO:0031053

Locations

nuclear dicing bodyGO:0010445

SE has no gene review yet, so this MF is asserted here from the in vitro reconstitution (PMID:18632569) rather than from a reviewed core function. SE also acts in pre-mRNA splicing with the cap-binding complex; that role is outside this module.

Part 2: 3'-terminal 2'-O-methylation of the miRNA/miRNA* duplex

HEN1 2'-O-methylation of miRNA/miRNA* duplexesReactionhen1_methylation

HEN1 transfers a methyl group from S-adenosyl-L-methionine to the 2'-OH of the 3'-terminal ribose on both strands of the DCL1-generated duplex. HEN1 alone is sufficient for this reaction in vitro. Without the methyl mark, miRNAs are 3'-uridylated and trimmed, and their levels fall. HEN1 also methylates siRNA duplexes, so this step is shared with siRNA pathways.

Annotons

HEN1: small RNA 2'-O-ribose methyltransferase

hen1_mtase

Participant: Gene Product: HEN1

Gene Product:

HEN1UniProtKB:Q9C5Q8

Function

small RNA 2'-O-ribose methyltransferase activityGO:0090486

Processes

miRNA processingGO:0035196

Locations

nucleusGO:0005634

Only the nuclear location is asserted. The HEN1 gene review leaves its cytoplasm rows UNDECIDED.

Part 3: guide-strand loading into AGO1 and miRISC target slicing

AGO1 loading and miRNA-guided target slicing (miRISC)Protein Complexago1_mirisc

The methylated miRNA strand is loaded into AGO1. AGO1 binds miRNAs, trans-acting siRNAs and transgene siRNAs but excludes 24-nt heterochromatic siRNAs. Loaded AGO1 cleaves complementary target mRNAs with its PIWI-domain catalytic centre. The active slicer is about 150 kDa, probably AGO1 with its guide RNA and no other protein, rather than a large animal-type RISC. AGO1 also represses target translation at the endoplasmic reticulum (modelled as a separate annoton). HYL1 and AGO1 interact in BiFC, which suggests that strand selection may be coupled to the microprocessor, but the loading mechanism is not modelled here.

RISC complexGO:0016442

Annotons

AGO1: miRNA guide binding (loaded effector)

ago1_guide_binding

Participant: Gene Product: AGO1

Gene Product:

AGO1UniProtKB:O04379

Function

miRNA bindingGO:0035198

Processes

miRNA-mediated post-transcriptional gene silencingGO:0035195

miRNA binding is retained (ACCEPT) but is not listed as a core MF in the AGO1 gene review, whose core MFs are the slicer, siRNA binding and translational repression. It is modelled here because this annoton is the loading step itself.

AGO1: miRNA-guided target RNA endonuclease (slicer)

ago1_slicer

Participant: Gene Product: AGO1

Gene Product:

AGO1UniProtKB:O04379

Function

endoribonuclease activity, cleaving miRNA-paired mRNAGO:0090624

Processes

miRNA-mediated post-transcriptional gene silencingGO:0035195

Locations

cytoplasmGO:0005737

GO:0090624 is a child of RNA endonuclease activity (GO:0004521), the term the AGO1 gene review uses as its core MF. The more specific term is used here because the annoton's evidence is miRNA-guided cleavage of a paired target mRNA; siRNA-guided slicing by AGO1 is outside this module.

AGO1: miRNA-guided translational repression at the endoplasmic reticulum

ago1_translational_repression

Participant: Gene Product: AGO1

Gene Product:

AGO1UniProtKB:O04379

Function

translation repressor activityGO:0030371

Processes

miRNA-mediated gene silencing by inhibition of translationGO:0035278

Locations

endoplasmic reticulumGO:0005783

Modelled because the module's scope includes the action of the loaded miRISC, and the AGO1 gene review treats translational repression as a core function alongside slicing. The ER membrane protein AMP1, which is also required for this repression, has no Arabidopsis gene review and is not modelled.