Cooperative recruitment of RDR6 by SGS3 and SDE5 during small interfering RNA amplification in Arabidopsis

Small interfering RNAs (siRNAs) are often amplified from transcripts cleaved by RNA-induced silencing complexes (RISCs) containing a small RNA (sRNA) and an Argonaute protein. Amplified siRNAs, termed secondary siRNAs, are important for reinforcement of target repression. In plants, target cleavage by RISCs containing 22-nucleotide (nt) sRNA and Argonaute 1 (AGO1) triggers siRNA amplification. In this pathway, the cleavage fragment is converted into double-stranded RNA (dsRNA) by RNA-dependent RNA polymerase 6 (RDR6), and the dsRNA is processed into siRNAs by Dicer-like proteins. Because nonspecific RDR6 recruitment causes nontarget siRNA production, it is critical that RDR6 is specifically recruited to the target RNA that serves as a template for dsRNA formation. Previous studies showed that Suppressor of Gene Silencing 3 (SGS3) binds and stabilizes 22-nt sRNA–containing AGO1 RISCs associated with cleaved target, but how RDR6 is recruited to targets cleaved by 22-nt sRNA–containing AGO1 RISCs remains unknown. Here, using cell-free extracts prepared from suspension-cultured Arabidopsis thaliana cells, we established an in vitro system for secondary siRNA production in which 22-nt siRNA–containing AGO1-RISCs but not 21-nt siRNA–containing AGO1-RISCs induce secondary siRNA production. In this system, addition of recombinant Silencing Defective 5 (SDE5) protein remarkably enhances secondary siRNA production. We show that RDR6 is recruited to a cleavage fragment by 22-nt siRNA–containing AGO1-RISCs in coordination with SGS3 and SDE5. The SGS3–SDE5–RDR6 multicomponent recognition system and the poly(A) tail inhibition may contribute to securing specificity of siRNA amplification.

Cell-free reconstitution reveals the molecular mechanisms for the initiation of secondary siRNA biogenesis in plants

Secondary small interfering RNA (siRNA) production, triggered by primary small RNA targeting, is critical for proper development and antiviral defense in many organisms. RNA-dependent RNA polymerase (RDR) is a key factor in this pathway. However, how RDR specifically converts the targets of primary small RNAs into double-stranded RNA (dsRNA) intermediates remains unclear. Here, we develop an in vitro system that allows for dissection of the molecular mechanisms underlying the production of trans-acting siRNAs, a class of plant secondary siRNAs that play roles in organ development and stress responses. We find that a combination of the dsRNA-binding protein, SUPPRESSOR OF GENE SILENCING3; the putative nuclear RNA export factor, SILENCING DEFECTIVE5, primary small RNA, and Argonaute is required for physical recruitment of RDR6 to target RNAs. dsRNA synthesis by RDR6 is greatly enhanced by the removal of the poly(A) tail, which can be achieved by the cleavage at a second small RNA-binding site bearing appropriate mismatches. Importantly, when the complementarity of the base pairing at the second target site is too strong, the small RNA–Argonaute complex remains at the cleavage site, thereby blocking the initiation of dsRNA synthesis by RDR6. Our data highlight the light and dark sides of double small RNA targeting in the secondary siRNA biogenesis.

Nuclear and cytoplasmic RNA exosomes and PELOTA1 prevent miRNA-induced secondary siRNA production in Arabidopsis

Amplification of short interfering RNA (siRNAs) via RNA dependent RNA Polymerases (RdRPs) is of fundamental importance in RNA silencing. In plants, silencing by microRNAs (miRNAs) generally does not lead to engagement of RdRPs, in part thanks to an as yet poorly understood activity of the cytoplasmic exosome adaptor SKI2. Here, we show that mutation of the cytoplasmic exosome subunit RRP45B results in siRNA production very similar to what is observed in ski2 mutants. Furthermore, loss of the nuclear exosome adaptor HEN2 leads to secondary siRNA production from miRNA targets largely distinct from those producing siRNAs in ski2. Importantly, mutation of the Release Factor paralogue PELOTA1 required for subunit dissociation of stalled ribosomes causes siRNA production from miRNA targets overlapping with, but distinct from, those affected in ski2 and rrp45b mutants. We also show that miRNA-induced illicit secondary siRNA production correlates with miRNA levels rather than accumulation of stable 5'-cleavage fragments. We propose that stalled RNA-induced Silencing Complex (RISC) and ribosomes, but not stable target mRNA cleavage fragments released from RISC, trigger secondary siRNA production, and that the exosome limits siRNA amplification by reducing RISC dwell time on miRNA target mRNAs while PELOTA1 does so by reducing ribosome stalling.

Ribosome stalling caused by the Argonaute-miRNA-SGS3 complex regulates production of secondary siRNA biogenesis in plants

The path of ribosomes on mRNAs can be impeded by various obstacles. One such example is halting of ribosome movement by microRNAs, though the exact mechanism and physiological role remain unclear. Here, we find that ribosome stalling caused by the Argonaute-miRNA-SGS3 complex regulates production of secondary siRNA biogenesis in plants. We show that the double-stranded RNA-binding protein, SGS3, directly interacts with the 3′ end of the microRNA-Argonaute complex, resulting in ribosome stalling. Strikingly, microRNA-mediated ribosome stalling enhances production of secondary small interfering RNAs (siRNAs) from target mRNAs. Our results uncover a previously uncharacterized role for paused ribosomes in regulation of small RNA function that may have broad biological implications across the plant kingdom.

Genome-wide CRISPR screening identifies new regulators of glycoprotein secretion

Background: The fundamental process of protein secretion from eukaryotic cells has been well described for many years, yet gaps in our understanding of how this process is regulated remain. Methods: With the aim of identifying novel genes involved in the secretion of glycoproteins, we used a screening pipeline consisting of a pooled genome-wide CRISPR screen, followed by secondary siRNA screening of the hits to identify and validate several novel regulators of protein secretion. Results: We present approximately 50 novel genes not previously associated with protein secretion, many of which also had an effect on the structure of the Golgi apparatus. We further studied a small selection of hits to investigate their subcellular localisation. One of these, GPR161, is a novel Golgi-resident protein that we propose maintains Golgi structure via an interaction with golgin A5. Conclusions: This study has identified new factors for protein secretion involved in Golgi homeostasis.

Genome-wide CRISPR screening identifies new regulators of glycoprotein secretion

Background: The fundamental process of protein secretion from eukaryotic cells has been well described for many years, yet gaps in our understanding of how this process is regulated remain. Methods: With the aim of identifying novel genes involved in the secretion of glycoproteins, we used a screening pipeline consisting of a pooled genome-wide CRISPR screen, followed by secondary siRNA screening of the hits to identify and validate several novel regulators of protein secretion. Results: We present approximately 50 novel genes not previously associated with protein secretion, many of which also had an effect on the structure of the Golgi apparatus. We further studied a small selection of hits to investigate their subcellular localisation. One of these, GPR161, is a novel Golgi-resident protein that we propose maintains Golgi structure via an interaction with golgin A5. Conclusions: This study has identified new factors for protein secretion involved in Golgi homeostasis.

Genome-wide CRISPR screening identifies new regulators of glycoprotein secretion

ABSTRACTThe fundamental process of protein secretion from eukaryotic cells has been well described for many years, yet gaps in our understanding of how this process is regulated remain. With the aim of identifying novel genes involved in the secretion of glycoproteins, we used a screening pipeline consisting of a pooled genome-wide CRISPR screen followed by secondary siRNA screening of the hits to identify and validate several novel regulators of protein secretion. We present approximately 50 novel genes not previously associated with protein secretion, many of which also had an effect on the structure of the Golgi apparatus. We further studied a small selection of hits to investigate their subcellular localisation. One of these, GPR161, is a novel Golgi-resident protein that we propose maintains Golgi structure via an interaction with golgin A5.

MicroRNAs from the parasitic plant Cuscuta campestris target host messenger RNAs

First paragraphDodders (Cuscuta spp.) are obligate parasitic plants that obtain water and nutrients from the stems of host plants via specialized feeding structures called haustoria. Dodder haustoria facilitate bi-directional movement of viruses, proteins, and mRNAs between host and parasite1, but the functional effects of these movements are not clear. Here we show that C. campestris haustoria accumulate high levels of many novel microRNAs (miRNAs) while parasitizing Arabidopsis thaliana hosts. Many of these miRNAs are 22 nts long, a usually rare size of plant miRNA associated with amplification of target silencing through secondary small interfering RNA (siRNA) production2. Several A. thaliana mRNAs are targeted by C. campestris 22 nt miRNAs during parasitism, resulting in mRNA cleavage, secondary siRNA production, and decreased mRNA accumulation levels. Hosts with mutations in two of the targets supported significantly higher growth of C. campestris. Homologs of target mRNAs from diverse plants also have predicted target sites to induced C. campestris miRNAs, and the same miRNAs are expressed and active against host targets when C. campestris parasitizes a different host, Nicotiana benthamiana. These data show that C. campestris miRNAs act as trans-species regulators of host gene expression, and suggest that they may act as virulence factors during parasitism.