How intrinsically disordered proteins order plant gene silencing

Abstract

Intrinsically disordered proteins (IDPs) and proteins with intrinsically disordered regions (IDRs) possess low sequence complexity of amino acids and display non-globular tertiary structures. They can act as scaffolds, form regulatory hubs, or trigger biomolecular condensation to control diverse aspects of biology. Emerging evidence has recently implicated critical roles of IDPs and IDR-contained proteins in nuclear transcription and cytoplasmic post-transcriptional processes, among other molecular functions. We here summarize the concepts and organizing principles of IDPs. We then illustrate recent progress in understanding the roles of key IDPs in machineries that regulate transcriptional and post-transcriptional gene silencing (PTGS) in plants, aiming at highlighting new modes of action of IDPs in controlling biological processes.

Keywords: gene silencing; intrinsically disordered protein (IDP); intrinsically disordered region (IDR); liquid–liquid phase separation; miRNA; siRNA.

Figure 1:. Interaction principles of IDPs and predicted fraction of IDPs with LLPS capacity in Arabidopsis and rice.

(A) IDPs are involved in macromolecule complex assembly or condensation via multivalent or transient interaction with their partners. IDPs recruit their partners, such as nucleic acids and folded proteins, via driving forces of electrostatic or hydrophobic interactions, to trigger macromolecule complex assembly, disassembly, condensation, and/or aggregation. The interactions are mostly multivalent and transient, determined by thermodynamic factors of IDPs or their partners, such as protein concentration, ATP level, temperature, balance of net charge, cellular physiological conditions, PTMs, etc. The features of IDPs and their assembly/condensation confer their roles in cellular compartmentation, conformational change, accessibility to PTM modifications, sequestering and scavengers, etc., thus to fine tune reaction activities, storage, and/or cleavage of proteins. (B-E) Overlap of top 30% of proteins predicted from five computational analyses to retain LLPS capacity (B and C) and their GO enrichment (D and E). The top 30% of proteins to retain LLPS capacity were predicted by SaPS, PdPS, catGRANULE, PLAAC, and PScore in Arabidopsis (B) and rice (C). GO enrichment for the 1036 and 252 overlapped proteins retain LLPS capacity in Arabidopsis (D) and rice (E). BP: Biological Process; CC: Cellular Component; MF: Molecular Function.

Figure 2:. IDPs regulate transcription and transcriptional silencing in plants.

Chromatin accessibility regulates gene transcription activity. (a) Chromatin compaction is regulated by condensation of histone variant H2B.8, and diverse regulators of histone and DNA modifications. (b) In the 24-nt siRNA-mediated RdDM pathway, RDR2 physically interacts with NRPD1 of the RNA Pol IV, and catalyzes RNA Pol IV transcripts into dsRNAs of ~30 bp. The dsRNAs are further cleaved into 24-nt siRNAs by DCL3, and loaded into AGO4 to target loci via base-pairing with Pol V transcripts, thus to recruit DNA methyltransferase DRM2 and histone-modifying enzymes to facilitate silencing of the target loci. In the process, RDR2, DCL3, AGO4 co-localize with the Cajal bodies, which are the sites for efficient RNP complex assembly. (c) The 24-nt siRNA-loaded AGO4 colocalizes with NRPD1b of the RNA Pol IV in the so-called AGO4-NRPD1b bodies which are proposed to facilitate silencing of the target loci by base-pairing with Pol V-derived non-coding RNA transcripts, and the recruitment of DRM2 and histone-modifying enzymes. (d) LHP1 and EMB1579 are IDPs to drive the assembly of PRC1/PRC2 into PcG bodies which trigger histone methylation and chromatin compaction. (e and f) The transcription of FLC is not only regulated by histone and DNA modifications, but also by FRI condensates (e) and 3’ polyadenylation condensates (f). FRI itself is an IDP and undergoes phase separation, which in turn sequesters FRI from FLC promoter to repress FRI activation of FLC transcription. FRI condensation is regulated by FLC antisense transcript COOLAIR. Meanwhile, FLL2 would act as an IDP to trigger the IDP FCA to undergo LLPS and assemble into 3’ polyadenylation condensates, thus to regulate 3’ polyadenylation and stability of COOLAIR. Intriguingly, the RNA Pol II CTD has an IDR and its phosphorylation status triggers the dynamics and exchanges of transcriptional condensates (g), adding to the complexity of transcriptional regulation. Abbreviations: AGO4, ARGONAUTE4; BMI1, also known as DRIP1, DREB2A-INTERACTING PROTEIN 1; CLF, CURLY LEAF; DCL3, DICER-LIKE 3; EMB1579, EMBRYO DEFECTIVE 1579; EMF1, EMBRYONIC FLOWER 1; EMF2, EMBRYONIC FLOWER 2; FCA, FIE, FERTILIZATION INDEPENDENT ENDOSPERM; FLOWERING CONTROL LOCUS A; FLL2, FLX-LIKE 2; FRI, FRIGIDA; LHP1, LIKE HETEROCHROMATIN PROTEIN 1; MSI1, MULTICOPY SUPRESSOR OF IRA1; PRC1, POLYCOMB REPRESSIVE COMPLEX 1; PRC2, POLYCOMB REPRESSIVE COMPLEX 2; RDR2, RNA-DEPENDENT RNA POLYMERASE 2; SWN, SWINGER; VRN2, VERNALIZATION2.

Figure 3:. IDPs in the regulation of miRNA pathway in plants.

(A) The microprocessor core is composed of DCL, HYL, and SE. As an IDP, SE plays multifaced roles in diverse macro-complex assembly, especially in the recruitment of HYL1, DCL1, and other co-factors of the microprocessor. (a) Pri-miRNAs transcribed by RNA Pol II are processed co-transcriptionally by the microprocessor. (b) SE phase separation would trigger the recruitment of HYL1, DCL1, and other co-factors into the microprocessor, thus for efficient production of miRNAs. Phase separation capacity of SE might be regulated by phosphorylation catalyzed by PRP4KA, and phosphorylated SE is subjected to 20S proteasome cleavage. Intriguingly, one family of IDPs, SAID1/2 are recruited by SE to undergo phase separation, whereas they can sequester pri-miRNAs from SE, impede microprocessor activity, and trigger SE phosphorylation and degradation, thus to constrain SE homeostasis and miRNA from overproduction. The RNA helicase RH6/8/12 are also IDPs that act to modulate SE phase separation. The peptidyl-prolyl isomerase CYP71 which catalyzes cis-trans isomerization of SE also acts to promote SE condensation and certain miRNA production. (c) After production, mature miRNAs are associated with HYL1 and subsequently routed into a hypothesized loading complex recruited by the IDP CARP9. As an IDP, CARP9 could recruit and interact with HYL1 and AGO1, thus facilitating the loading of miRNAs into RISCs to fulfil mRNA cleavage or translation inhibition. (B) An AlphaFold2 modeling predicts conformational change of SE and SAID1/2 upon binding with pri-miRNAs. SIAD1 and SAID2 monomers are less-structured IDPs, upon interaction with the IDP SE and pri-miRNAs, all proteins are predicted to change their conformation and become structured.

Figure 4:. A proposed model for SGS condensate in plant 21–22 nt siRNA biogenesis in PTGS.

SGS3 is an IDP and retains capacity to undergo phase separation. RDR6, ssRNAs, and other siRNA processing components, are recruited by SGS3 to form 21–22nt siRNA bodies in the cytoplasm. Phosphorylation of SGS3 (at Ser36 and Ser37) would impair its phase separation and siRNA body assembly. The WD-40 protein FVE triggers SGS3 dimerization and its binding with ssRNA substrate. Subsequently, ssRNAs are converted to dsRNAs by RDR6, and the dsRNAs are relayed to DCL2/4-DRB4 complexes for efficient cleavage into siRNA by DCL2/4, which are hypothesized to be occurred in the siRNA bodies. The siRNAs are subsequently loaded into corresponding AGO proteins and direct cleavage and silencing of their RNA targets.