The Nuclear Ribonucleoprotein SmD1 Interplays with Splicing, RNA Quality Control, and Posttranscriptional Gene Silencing in Arabidopsis.

RNA quality control (RQC) eliminates aberrant RNAs based on their atypical structure, whereas posttranscriptional gene silencing (PTGS) eliminates both aberrant and functional RNAs through the sequence-specific action of short interfering RNAs (siRNAs). The Arabidopsis thaliana mutant smd1b was identified in a genetic screen for PTGS deficiency, revealing the involvement of SmD1, a component of the Smith (Sm) complex, in PTGS. The smd1a and smd1b single mutants are viable, but the smd1a smd1b double mutant is embryo-lethal, indicating that SmD1 function is essential. SmD1b resides in nucleoli and nucleoplasmic speckles, colocalizing with the splicing-related factor SR34. Consistent with this, the smd1b mutant exhibits intron retention at certain endogenous mRNAs. SmD1 binds to RNAs transcribed from silenced transgenes but not nonsilenced ones, indicating a direct role in PTGS. Yet, mutations in the RQC factors UPFRAMESHIFT3, EXORIBONUCLEASE2 (XRN2), XRN3, and XRN4 restore PTGS in smd1b, indicating that SmD1 is not essential for but rather facilitates PTGS. Moreover, the smd1b mtr4 double mutant is embryo-lethal, suggesting that SmD1 is essential for mRNA TRANSPORT REGULATOR4-dependent RQC. These results indicate that SmD1 interplays with splicing, RQC, and PTGS. We propose that SmD1 facilitates PTGS by protecting transgene-derived aberrant RNAs from degradation by RQC in the nucleus, allowing sufficient amounts to enter cytoplasmic siRNA bodies to activate PTGS.

Cytoplasmic and nuclear quality control and turnover of single-stranded RNA modulate post-transcriptional gene silencing in plants.

Eukaryotic RNA quality control (RQC) uses both endonucleolytic and exonucleolytic degradation to eliminate dysfunctional RNAs. In addition, endogenous and exogenous RNAs are degraded through post-transcriptional gene silencing (PTGS), which is triggered by the production of double-stranded (ds)RNAs and proceeds through short-interfering (si)RNA-directed ARGONAUTE-mediated endonucleolytic cleavage. Compromising cytoplasmic or nuclear 5'-3' exoribonuclease function enhances sense-transgene (S)-PTGS in Arabidopsis, suggesting that these pathways compete for similar RNA substrates. Here, we show that impairing nonsense-mediated decay, deadenylation or exosome activity enhanced S-PTGS, which requires host RNA-dependent RNA polymerase 6 (RDR6/SGS2/SDE1) and SUPPRESSOR OF GENE SILENCING 3 (SGS3) for the transformation of single-stranded RNA into dsRNA to trigger PTGS. However, these RQC mutations had no effect on inverted-repeat-PTGS, which directly produces hairpin dsRNA through transcription. Moreover, we show that these RQC factors are nuclear and cytoplasmic and are found in two RNA degradation foci in the cytoplasm: siRNA-bodies and processing-bodies. We propose a model of single-stranded RNA tug-of-war between RQC and S-PTGS that ensures the correct partitioning of RNA substrates among these RNA degradation pathways.

Long noncoding RNA modulates alternative splicing regulators in Arabidopsis.

Alternative splicing (AS) of pre-mRNA represents a major mechanism underlying increased transcriptome and proteome complexity. Here, we show that the nuclear speckle RNA-binding protein (NSR) and the AS competitor long noncoding RNA (or ASCO-lncRNA) constitute an AS regulatory module. AtNSR-GFP translational fusions are expressed in primary and lateral root (LR) meristems. Double Atnsr mutants and ASCO overexpressors exhibit an altered ability to form LRs after auxin treatment. Interestingly, auxin induces a major change in AS patterns of many genes, a response largely dependent on NSRs. RNA immunoprecipitation assays demonstrate that AtNSRs interact not only with their alternatively spliced mRNA targets but also with the ASCO-RNA in vivo. The ASCO-RNA displaces an AS target from an NSR-containing complex in vitro. Expression of ASCO-RNA in Arabidopsis affects the splicing patterns of several NSR-regulated mRNA targets. Hence, lncRNA can hijack nuclear AS regulators to modulate AS patterns during development.

Independent activity of the homologous small regulatory RNAs AbcR1 and AbcR2 in the legume symbiont Sinorhizobium meliloti.

The legume symbiont Sinorhizobium meliloti expresses a plethora of small noncoding RNAs (sRNAs) whose function is mostly unknown. Here, we have functionally characterized two tandemly encoded S. meliloti Rm1021 sRNAs that are similar in sequence and structure. Homologous sRNAs (designated AbcR1 and AbcR2) have been shown to regulate several ABC transporters in the related α-proteobacteria Agrobacterium tumefaciens and Brucella abortus. In Rm1021, AbcR1 and AbcR2 exhibit divergent unlinked regulation and are stabilized by the RNA chaperone Hfq. AbcR1 is transcribed in actively dividing bacteria, either in culture, rhizosphere or within the invasion zone of mature alfalfa nodules. Conversely, AbcR2 expression is induced upon entry into stationary phase and under abiotic stress. Only deletion of AbcR1 resulted into a discrete growth delay in rich medium, but both are dispensable for symbiosis. Periplasmic proteome profiling revealed down-regulation of the branched-chain amino acid binding protein LivK by AbcR1, but not by AbcR2. A double-plasmid reporter assay confirmed the predicted specific targeting of the 5'-untranslated region of the livK mRNA by AbcR1 in vivo. Our findings provide evidences of independent regulatory functions of these sRNAs, probably to fine-tune nutrient uptake in free-living and undifferentiated symbiotic rhizobia.

Dual RNAs in plants.

Plants have remarkable developmental plasticity, and the same genotype can result in different phenotypes depending on environmental variation. Indeed, abiotic stresses or biotic interactions affect organogenesis and post-embryonic growth and significantly affect gene regulation. The large diversity of non-protein-coding RNAs (npcRNAs) and genes containing only short open reading frames that are expressed during plant growth and development, contribute to the regulation of gene expression. Certain npcRNAs code for oligopeptides and may possess additional biological activity linked to the RNA moiety. The ENOD40 gene is a dual RNA that is activated during a symbiotic interaction leading to root nodule organogenesis. Both the oligopeptides encoded by ENOD40 and the structured regions of the ENOD40 RNA have been shown to interact with different proteins in the cell to control enzymatic activities or induce the relocalisation of ribonucleoproteins, respectively. Other npcRNAs encode for small signalling peptides or are the precursors of small RNAs involved in post-transcriptional or transcriptional gene silencing. They may have RNA-related activities or encode peptides (or even larger proteins), and therefore act as dual RNAs. In addition, long natural antisense RNAs with a coding function and a regulatory RNA-mediated action that are expressed in response to abiotic stress in plants have been identified. In certain cases, these RNAs lead to the synthesis of nat-siRNAs, that are small RNAs derived from the overlapping double-stranded RNA region of natural antisense RNAs, which facilitates the silencing of complementary mRNAs. Finally, the advent of deep sequencing technologies has identified a large number of non-protein-coding RNAs in plants, which could be a large reservoir for dual RNAs.

Non-protein-coding RNAs and their interacting RNA-binding proteins in the plant cell nucleus.

The complex responses of eukaryotic cells to external factors are governed by several transcriptional and post-transcriptional processes. Several of them occur in the nucleus and have been linked to the action of non-protein-coding RNAs (or npcRNAs), both long and small npcRNAs, that recently emerged as major regulators of gene expression. Regulatory npcRNAs acting in the nucleus include silencing-related RNAs, intergenic npcRNAs, natural antisense RNAs, and other aberrant RNAs resulting from the interplay between global transcription and RNA processing activities (such as Dicers and RNA-dependent polymerases). Generally, the resulting npcRNAs exert their regulatory effects through interactions with RNA-binding proteins (or RBPs) within ribonucleoprotein particles (or RNPs). A large group of RBPs are implicated in the silencing machinery through small interfering RNAs (siRNAs) and their localization suggests that several act in the nucleus to trigger epigenetic and chromatin changes at a whole-genome scale. Other nuclear RBPs interact with npcRNAs and change their localization. In the fission yeast, the RNA-binding Mei2p protein, playing pivotal roles in meiosis, interact with a meiotic npcRNA involved in its nuclear re-localization. Related processes have been identified in plants and the ENOD40 npcRNA was shown to re-localize a nuclear-speckle RBP from the nucleus to the cytoplasm in Medicago truncatula. Plant RBPs have been also implicated in RNA-mediated chromatin silencing in the FLC locus through interaction with specific antisense transcripts. In this review, we discuss the interactions between RBPs and npcRNAs in the context of nuclear-related processes and their implication in plant development and stress responses. We propose that these interactions may add a regulatory layer that modulates the interactions between the nuclear genome and the environment and, consequently, control plant developmental plasticity.