A viral noncoding RNA generated by cis-element-mediated protection against 5'->3' RNA decay represses both cap-independent and cap-dependent translation

Positive-strand RNA viruses use diverse mechanisms to regulate viral and host gene expression for ensuring their efficient proliferation or persistence in the host. We found that a small viral noncoding RNA (0.4 kb), named SR1f, accumulated in Red clover necrotic mosaic virus (RCNMV)-infected plants and protoplasts and was packaged into virions. The genome of RCNMV consists of two positive-strand RNAs, RNA1 and RNA2. SR1f was generated from the 3′ untranslated region (UTR) of RNA1, which contains RNA elements essential for both cap-independent translation and negative-strand RNA synthesis. A 58-nucleotide sequence in the 3′ UTR of RNA1 (Seq1f58) was necessary and sufficient for the generation of SR1f. SR1f was neither a subgenomic RNA nor a defective RNA replicon but a stable degradation product generated by Seq1f58-mediated protection against 5′→3′ decay. SR1f efficiently suppressed both cap-independent and cap-dependent translation both in vitro and in vivo. SR1f trans inhibited negative-strand RNA synthesis of RCNMV genomic RNAs via repression of replicase protein production but not via competition of replicase proteins in vitro. RCNMV seems to use cellular enzymes to generate SR1f that might play a regulatory role in RCNMV infection. Our results also suggest that Seq1f58 is an RNA element that protects the 3′-side RNA sequences against 5′→3′ decay in plant cells as reported for the poly(G) tract and stable stem-loop structure in Saccharomyces cerevisiae.     

Implication of CcpN in the regulation of a novel untranslated RNA (SR1) in Bacillus subtilis

Antisense-RNAs have been investigated in detail over the past 20 years as the principal regulators in accessory DNA elements such as plasmids, phages and transposons. However, only a few examples of chromosomally encoded bacterial antisense RNAs were known. Meanwhile, approximately 70 small non-coding RNAs from the Escherichia coli genome have been found, the functions of the majority of which remain to be elucidated. Only one systematic search has been performed for Gram-positive bacteria, so far. Here, we report the identification of a novel small (205 nt) non-translated RNA--SR1--encoded in the Bacillus subtilis genome. SR1 was predicted by a computational approach and verified by Northern blotting. Knockout or overexpression of SR1 did not affect growth. SR1 was derepressed under conditions of gluconeogenesis, but repressed under glycolytic conditions. Two regulatory levels could be identified, one involving CcpA, the second, more important, involving the recently identified regulator CcpN.     

One antitoxin—two functions: SR4 controls toxin mRNA decay and translation

Type I toxin–antitoxin systems encoded on bacterial chromosomes became the focus of research during the past years. However, little is known in terms of structural requirements, kinetics of interaction with their targets and regulatory mechanisms of the antitoxin RNAs. Here, we present a combined in vitro and in vivo analysis of the bsrG/SR4 type I toxin–antitoxin system from Bacillus subtilis. The secondary structures of SR4 and bsrG mRNA and of the SR4/bsrG RNA complex were determined, apparent binding rate constants calculated and functional segments required for complex formation narrowed down. The initial contact between SR4 and its target was shown to involve the SR4 terminator loop and loop 3 of bsrG mRNA. Additionally, a contribution of the stem of SR4 stem-loop 3 to target binding was found. On SR4/bsrG complex formation, a 4 bp double-stranded region sequestering the bsrG Shine Dalgarno (SD) sequence was extended to 8 bp. Experimental evidence was obtained that this extended region caused translation inhibition of bsrG mRNA. Therefore, we conclude that SR4 does not only promote degradation of the toxin mRNA but also additionally inhibit its translation. This is the first case of a dual-acting antitoxin RNA.     

Interactions among SR proteins, an exonic splicing enhancer, and a lentivirus Rev protein regulate alternative splicing.

We examine here the roles of cellular splicing factors and virus regulatory proteins in coordinately regulating alternative splicing of the tat/rev mRNA of equine infectious anemia virus (EIAV). This bicistronic mRNA contains four exons; exons 1 and 2 encode Tat, and exons 3 and 4 encode Rev. In the absence of Rev expression, the four-exon mRNA is synthesized exclusively, but when Rev is expressed, exon 3 is skipped to produce an mRNA that contains only exons 1, 2, and 4. We identify a purine-rich exonic splicing enhancer (ESE) in exon 3 that promotes exon inclusion. Similar to other cellular ESEs that have been identified by other laboratories, the EIAV ESE interacted specifically with SR proteins, a group of serine/arginine-rich splicing factors that function in constitutive and alternative mRNA splicing. Substitution of purines with pyrimidines in the ESE resulted in a switch from exon inclusion to exon skipping in vivo and abolished binding of SR proteins in vitro. Exon skipping was also induced by expression of EIAV Rev. We show that Rev binds to exon 3 RNA in vitro, and while the precise determinants have not been mapped, Rev function in vivo and RNA binding in vitro indicate that the RNA element necessary for Rev responsiveness overlaps or is adjacent to the ESE. We suggest that EIAV Rev promotes exon skipping by interfering with SR protein interactions with RNA or with other splicing factors.     

Plants Encode a General siRNA Suppressor That Is Induced and Suppressed by Viruses

Small RNAs play essential regulatory roles in genome stability, development, and responses to biotic and abiotic stresses in most eukaryotes. In plants, the RNaseIII enzyme DICER-LIKE1 (DCL1) produces miRNAs, whereas DCL2, DCL3, and DCL4 produce various size classes of siRNAs. Plants also encode RNASE THREE-LIKE (RTL) enzymes that lack DCL-specific domains and whose function is largely unknown. We found that virus infection induces RTL1 expression, suggesting that this enzyme could play a role in plant–virus interaction. To first investigate the biochemical activity of RTL1 independent of virus infection, small RNAs were sequenced from transgenic plants constitutively expressing RTL1. These plants lacked almost all DCL2-, DCL3-, and DCL4-dependent small RNAs, indicating that RTL1 is a general suppressor of plant siRNA pathways. In vivo and in vitro assays revealed that RTL1 prevents siRNA production by cleaving dsRNA prior to DCL2-, DCL3-, and DCL4-processing. The substrate of RTL1 cleavage is likely long-perfect (or near-perfect) dsRNA, consistent with the RTL1-insensitivity of miRNAs, which derive from DCL1-processing of short-imperfect dsRNA. Virus infection induces RTL1 mRNA accumulation, but viral proteins that suppress RNA silencing inhibit RTL1 activity, suggesting that RTL1 has evolved as an inducible antiviral defense that could target dsRNA intermediates of viral replication, but that a broad range of viruses counteract RTL1 using the same protein toolbox used to inhibit antiviral RNA silencing. Together, these results reveal yet another level of complexity in the evolutionary battle between viruses and plant defenses.     

Control of translation by the 5'- and 3'-terminal regions of the dengue virus genome

The genomic RNAs of flaviviruses such as dengue virus (DEN) have a 5' m7GpppN cap like those of cellular mRNAs but lack a 3' poly(A) tail. We have studied the contributions to translational expression of 5'- and 3'-terminal regions of the DEN serotype 2 genome by using luciferase reporter mRNAs transfected into Vero cells. DCLD RNA contained the entire DEN 5' and 3' untranslated regions (UTRs), as well as the first 36 codons of the capsid coding region fused to the luciferase reporter gene. Capped DCLD RNA was as efficiently translated in Vero cells as capped GLGpA RNA, a reporter with UTRs from the highly expressed alpha-globin mRNA and a 72-residue poly(A) tail. Analogous reporter RNAs with regulatory sequences from West Nile and Sindbis viruses were also strongly expressed. Although capped DCLD RNA was expressed much more efficiently than its uncapped form, uncapped DCLD RNA was translated 6 to 12 times more efficiently than uncapped RNAs with UTRs from globin mRNA. The 5' cap and DEN 3' UTR were the main sources of the translational efficiency of DCLD RNA, and they acted synergistically in enhancing translation. The DEN 3' UTR increased mRNA stability, although this effect was considerably weaker than the enhancement of translational efficiency. The DEN 3' UTR thus has translational regulatory properties similar to those of a poly(A) tail. Its translation-enhancing effect was observed for RNAs with globin or DEN 5' sequences, indicating no codependency between viral 5' and 3' sequences. Deletion studies showed that translational enhancement provided by the DEN 3' UTR is attributable to the cumulative contributions of several conserved elements, as well as a nonconserved domain adjacent to the stop codon. One of the conserved elements was the conserved sequence (CS) CS1 that is complementary to cCS1 present in the 5' end of the DEN polyprotein open reading frame. Complementarity between CS1 and cCS1 was not required for efficient translation.     

RNA在RNA剪接中的功能：从催化到调控

对非编码RNA功能的认识是后基因组时代的一个研究焦点,本文主要介绍非编码RNA在RNA剪接中的催化和调控功能。在RNA加工过程中,三大类内含子的剪接都是由RNA成员主导。其中Ⅰ型和Ⅱ型内含子能催化自身的切除和外显子连接反应;而核mRNA内含子的剪接则由剪接体里的小核RNA主导。Ⅰ型和Ⅱ型内含子存在于细菌、低等真核细胞和植物的细胞器内;而真核细胞的核编码蛋白质基因内全部是核mRNA内含子,并且其数目随生物体的复杂性而显著升高。一个多内含子前体mRNA通过选择性剪接产生多种,甚至上万种不同的mRNA和蛋白质,对蛋白质组的复杂度和时空表达调控至关重要。选择性剪接调控由剪接调控蛋白特异识别和结合前体mRNA里所富含的顺式RNA调控元件完成的;系统认识这两者之间的对应关系是揭示基因组表达调控网络的一把钥匙。     

Diversity of endogenous small non-coding RNAs in Oryza sativa

Small non-coding RNAs play important roles in regulating cell functions by controlling mRNA turnover and translational repression in eukaryotic cells. Here we isolated 162 endogenous small RNA molecules from Oryza sativa, which ranged from 16 to 35 nt in length. Further analysis indicated that they represented a diversity of small RNA molecules, including 17 microRNAs (miRNAs), 30 tiny non-coding RNAs (tncRNAs) and 20 repeat-associated small interfering RNAs (rasiRNAs). Among 17 miRNAs, 13 were novel miRNA candidates and their potential targets were important regulatory genes in the rice genome. We also found that a cluster of small RNAs, including many rasiRNAs, matched to a nuclear DNA fragment that evolutionarily derived from chloroplast. These results demonstrate clearly the existence of distinct types of small RNAs in rice and further suggest that small RNAs may control gene regulation through diverse mechanisms.     

Identification and Characterization of Argonaute Protein, Ago2 and Its Associated Small RNAs in Schistosoma japonicum

Background

The complex life cycle of the genus Schistosoma drives the parasites to employ subtle developmentally dependent gene regulatory machineries. Small non-coding RNAs (sncRNAs) are essential gene regulatory factors that, through their impact on mRNA and genome stability, control stage-specific gene expression. Abundant sncRNAs have been identified in this genus. However, their functionally associated partners, Argonaute family proteins, which are the key components of the RNA-induced silencing complex (RISC), have not yet been fully explored.

Methodology/Principal Findings

Two monoclonal antibodies (mAbs) specific to Schistosoma japonicum Argonaute protein Ago2 (SjAgo2), but not SjAgo1 and SjAgo3, were generated. Soluble adult worm antigen preparation (SWAP) was subjected to immunoprecipitation with the mAbs and the captured SjAgo2 protein was subsequently confirmed by Western blot and mass spectrometry (MS) analysis. The small RNA population associated with native SjAgo2 in adult parasites was extracted from the immunoprecipitated complex and subjected to library construction. High-through-put sequencing of these libraries yielded a total of ≈50 million high-quality reads. Classification of these small RNAs showed that endogenous siRNAs (endo-siRNAs) generated from transposable elements (TEs), especially from the subclasses of LINE and LTR, were prominent. Further bioinformatics analysis revealed that siRNAs derived from ten types of well-defined retrotransposons were dramatically enriched in the SjAgo2-specific libraries compared to small RNA libraries constructed with total small RNAs from separated adult worms. These results suggest that a key function of SjAgo2 is to maintain genome stability through suppressing the activities of retrotransposons.

Conclusions/Significance

In this study, we identified and characterized one of the three S. japonicum Argonautes, SjAgo2, and its associated small RNAs were found to be predominantly derived from particular classes of retrotransposons. Thus, a major function of SjAgo2 appears to associate with the maintenance of genome stability via suppression of retroelements. The data advance our understanding of the gene regulatory mechanisms in the blood fluke.     

Characterization of unique small RNA populations from rice grain

Small RNAs (approximately 20 to 24 nucleotides) function as naturally occurring molecules critical in developmental pathways in plants and animals. Here we analyze small RNA populations from mature rice grain and seedlings by pyrosequencing. Using a clustering algorithm to locate regions producing small RNAs, we classified hotspots of small RNA generation within the genome. Hotspots here are defined as 1 kb regions within which small RNAs are significantly overproduced relative to the rest of the genome. Hotspots were identified to facilitate characterization of different categories of small RNA regulatory elements. Included in the hotspots, we found known members of 23 miRNA families representing 92 genes, one trans acting siRNA (ta-siRNA) gene, novel siRNA-generating coding genes and phased siRNA generating genes. Interestingly, over 20% of the small RNA population in grain came from a single foldback structure, which generated eight phased 21-nt siRNAs. This is reminiscent of a newly arising miRNA derived from duplication of progenitor genes. Our results provide data identifying distinct populations of small RNAs, including phased small RNAs, in mature grain to facilitate characterization of small regulatory RNA expression in monocot species.     

lsm1 mutations impairing the ability of the Lsm1p-7p-Pat1p complex to preferentially bind to oligoadenylated RNA affect mRNA decay in vivo

The poly(A) tail is a crucial determinant in the control of both mRNA translation and decay. Poly(A) tail length dictates the triggering of the degradation of the message body in the major 5′ to 3′ and 3′ to 5′ mRNA decay pathways of eukaryotes. In the 5′ to 3′ pathway oligoadenylated but not polyadenylated mRNAs are selectively decapped in vivo, allowing their subsequent degradation by 5′ to 3′ exonucleolysis. The conserved Lsm1p-7p-Pat1p complex is required for normal rates of decapping in vivo, and the purified complex exhibits strong binding preference for oligoadenylated RNAs over polyadenylated or unadenylated RNAs in vitro. In the present study, we show that two lsm1 mutants produce mutant complexes that fail to exhibit such higher affinity for oligoadenylated RNA in vitro. Interestingly, these mutant complexes are normal with regard to their integrity and retain the characteristic RNA binding properties of the wild-type complex, namely, binding near the 3′-end of the RNA, having higher affinity for unadenylated RNAs that carry U-tracts near the 3′-end over those that do not and exhibiting similar affinities for unadenylated and polyadenylated RNAs. Yet, these lsm1 mutants exhibit a strong mRNA decay defect in vivo. These results underscore the importance of Lsm1p-7p-Pat1p complex–mRNA interaction for mRNA decay in vivo and imply that the oligo(A) tail mediated enhancement of such interaction is crucial in that process.