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PubMed2006

HEN1 recognizes 21-24 nt small RNA duplexes and deposits a methyl group onto the 2' OH of the 3' terminal nucleotide.

microRNAs (miRNAs) and small interfering RNAs (siRNAs) in plants bear a methyl group on the ribose of the 3' terminal nucleotide. We showed previously that the methylation of miRNAs and siRNAs requires the protein HEN1 in vivo and that purified HEN1 protein methylates miRNA/miRNA* duplexes in vitro. In this study, we show that HEN1 methylates both miRNA/miRNA* and siRNA/siRNA* duplexes in vitro with a preference for 21-24 nt RNA duplexes with 2 nt overhangs. We also demonstrate that HEN1 deposits the methyl group on to the 2' OH of the 3' terminal nucleotide. Among various modifications that can occur on the ribose of the terminal nucleotide, such as 2'-deoxy, 3'-deoxy, 2'-O-methyl and 3'-O-methyl, only 2'-O-methyl on a small RNA inhibits the activity of yeast poly(A) polymerase (PAP). These findings indicate that HEN1 specifically methylates miRNAs and siRNAs and implicate the importance of the 2'-O-methyl group in the biology of RNA silencing.

Escherichia coli Proteins↗
PubMed2006

A distinct small RNA pathway silences selfish genetic elements in the germline.

In the Drosophila germline, repeat-associated small interfering RNAs (rasiRNAs) ensure genomic stability by silencing endogenous selfish genetic elements such as retrotransposons and repetitive sequences. Whereas small interfering RNAs (siRNAs) derive from both the sense and antisense strands of their double-stranded RNA precursors, rasiRNAs arise mainly from the antisense strand. rasiRNA production appears not to require Dicer-1, which makes microRNAs (miRNAs), or Dicer-2, which makes siRNAs, and rasiRNAs lack the 2',3' hydroxy termini characteristic of animal siRNA and miRNA. Unlike siRNAs and miRNAs, rasiRNAs function through the Piwi, rather than the Ago, Argonaute protein subfamily. Our data suggest that rasiRNAs protect the fly germline through a silencing mechanism distinct from both the miRNA and RNA interference pathways.

Animals↗
PubMed1998

Characterization of a novel small RNA encoded by Dictyostelium discoideum mitochondrial DNA.

In this study, we analyzed a mitochondrial small (ms) RNA in Dictyostelium discoideum, which is 129 nucleotides long and has a GC content of only 22.5%. In the mitochondrial DNA, a single-copy gene (msr) for the ms RNA was located downstream of the gene for large-subunit rRNA. The location of msr was similar to that of the 5S rRNA gene in prokaryotes and chloroplasts, but clearly different from that in mitochondria of plants, liverwort and the chlorophycean alga Prototheca wikerhamii, in which small-subunit rRNA and 5S rRNA genes are closely linked. THe primary sequence of ms rRNA showed low homology with mitochondrial 5S rRNA from plants, liverwort and the chlorophycean alga, but the proposed secondary structure of ms RNA was similar to that of cytoplasmic 5S rRNA. In addition, ms RNA showed a highly conserved GAAC sequence in the same loop as in common 5S rRNA. However, ms RNA was detected mainly in the mitochondrial 25,000 x g supernatant fraction which was devoid of ribosomes. It is possible that ms RNA is an evolutionary derivative of mitochondrial 5S rRNA.

Animals↗
PubMed1997

Targeted deletion of sprA from the tobacco plastid genome indicates that the encoded small RNA is not essential for pre-16S rRNA maturation in plastids.

The small plastid RNA (spRNA) which includes a segment that is complementary to the pre-16S rRNA has been suggested to facilitate maturation of pre-16S rRNA in tobacco. To investigate the function of spRNA, the gene encoding it (sprA) was removed from the plastid genome using targeted gene deletion. We report here that deletion of sprA does not significantly affect pre-16S rRNA maturation, nor does it cause any obvious phenotype. Although the spRNA still may be involved in rRNA maturation, it is non-essential under normal growth conditions.

Gene Deletion↗
PubMed1989

Neutralization of small RNA viruses by antibodies and antiviral agents.

The 3-dimensional structures of a number of small animal RNA viruses are now known to near-atomic resolution. All these viruses have similar structures and have, in all probability, diverged from a common ancestral virus able to infect a variety of organisms. The structures have elucidated the site of attachment to host cell receptors and the mode of protection of this site against host immune pressure. An internal hydrophobic pocket in rhinoviruses is the site for binding of antiviral drugs that inhibit uncoating and can inhibit attachment in some viruses. The pocket is probably a functional necessity, and thus is a suitable target for well-designed antiviral agents in many viruses.

Antibodies, Viral↗
PubMed1989

Glucocorticoid-receptor complexes are associated with small RNA in vitro.

Identification of RNA associated with soluble glucocorticoid-receptor complexes of HeLa cells was performed by immunoprecipitation of receptor complexes with a monoclonal antibody raised against rat liver glucocorticoid receptor. Polyacrylamide gel electrophoresis of RNA extracted from immunoprecipitates of cytosolic complexes revealed the presence of eight RNA bands, consisting of 28S, 18S, and small RNAs, including 5.8S, 5S and tRNA. A comparison of RNA species immunoprecipitated by monoclonal anti-glucocorticoid receptor antibody and IgG purified from normal mouse serum showed that four small RNAs were preferentially recovered after immunoprecipitation with anti-glucocorticoid receptor antibody. When these species were analyzed on sequencing gels, their nucleotide lengths coincided with those of 7-3, 7S, U2, and U1 RNA. Immunoprecipitation of nuclear extracts containing glucocorticoid-receptor-RNA complexes showed that the same set of small RNAs was preferentially immunoprecipitated by anti-glucocorticoid receptor antibody. The four small RNAs we detected represented minor species in whole extracts, and their preferential immunoprecipitation by anti-glucocorticoid receptor antibody was prevented by removal of glucocorticoid-receptor complexes from HeLa cell extracts. We conclude that 7-3, 7S, U2, and U1 RNA are associated with glucocorticoid-receptor complexes in vitro, and hypothesize that post-transcriptional effects of glucocorticoids may in part be mediated through interaction of receptor complexes with these small RNAs.

Antibodies, Monoclonal↗
PubMed2000

Yeast exosome mutants accumulate 3'-extended polyadenylated forms of U4 small nuclear RNA and small nucleolar RNAs.

The exosome is a protein complex consisting of a variety of 3'-to-5' exonucleases that functions both in 3'-to-5' trimming of rRNA precursors and in 3'-to-5' degradation of mRNA. To determine additional exosome functions, we examined the processing of a variety of RNAs, including tRNAs, small nuclear RNAs (snRNAs), small nucleolar RNAs (snoRNAs), RNase P, RNase MRP, and SRP RNAs, and 5S rRNAs in mutants defective in either the core components of the exosome or in other proteins required for exosome function. These experiments led to three important conclusions. First, exosome mutants accumulate 3'-extended forms of the U4 snRNA and a wide variety of snoRNAs, including snoRNAs that are independently transcribed or intron derived. This finding suggests that the exosome functions in the 3' end processing of these species. Second, in exosome mutants, transcripts for U4 snRNA and independently transcribed snoRNAs accumulate as 3'-extended polyadenylated species, suggesting that the exosome is required to process these 3'-extended transcripts. Third, processing of 5.8S rRNA, snRNA, and snoRNA by the exosome is affected by mutations of the nuclear proteins Rrp6p and Mtr4p, whereas mRNA degradation by the exosome required Ski2p and was not affected by mutations in RRP6 or MTR4. This finding suggests that the cytoplasmic and nuclear forms of the exosome represent two functionally different complexes involved in distinct 3'-to-5' processing and degradation reactions.

DEAD-box RNA Helicases↗
PubMed1995

A small RNA acts as an antisilencer of the H-NS-silenced rcsA gene of Escherichia coli.

The regulation of capsular polysaccharide synthesis in Escherichia coli K-12 depends on the level of an unstable positive regulator, RcsA. The amount of RcsA protein is limited both by its rapid degradation by Lon, an ATP-dependent protease, and by its low level of synthesis. We have found that the low level of expression from the rcsA promoter is due to transcriptional silencing by the histone-like protein H-NS; this silencing is sensitive to both sequence and context in a region upstream of the -35 region of the promoter. A small (85-nt) RNA, DsrA, when overproduced, activates transcription of rcsA::lacZ fusions by counteracting H-NS silencing. DsrA RNA does not show any extended homology with the rcsA promoter or other sequenced regions of E. coli. Since the stimulation of rcsA transcription by this small RNA does not depend on any sequences from within the rcsA transcript, DsrA acts, either directly or indirectly, on rcsA transcription initiation.

ATP-Dependent Proteases↗
PubMed2002

Phylogeny of the Gyalectales and Ostropales (Ascomycota, Fungi): among and within order relationships based on nuclear ribosomal RNA small and large subunits.

Despite various morphological and anatomical similarities, the two orders Gyalectales (lichenized ascomycetes) and Ostropales (lichenized and non-lichenized ascomycetes) have been considered to be distantly related to each other and their position within the Ascomycota was unsettled. To estimate relationships within these groups and their respective phylogenenetic placement within the Ascomycota, we analyzed DNA sequences from the nuclear small and large subunit ribosomal RNA genes using Maximum Parsimony, Maximum Likelihood, and Bayesian statistics with Markov chain Monte Carlo algorithms. Support for internal branches estimated with bootstrap was compared to Bayesian posterior probabilities. We report here that the Ostropales, in their current circumscription, are paraphyletic, and that the Ostropales s.l. include the Gyalectales and Trapeliaceae. The Unitunicate Ascohymenials are redelineated to include the Ostropales s.l., as defined here, and the Baeomycetaceae. Dimerella and Coenogonium are congeneric, and Petractis thelotremella and P. hypoleuca are reunited with members of the genus Gyalecta. In addition to requiring less computational time, Bayesian inference of phylogeny recovered the same topology as a conventional heuristic search using Maximum Likelihood as the optimization criterion and seems superior to bootstrapping in estimating support for short internal branches.

Ascomycota↗
PubMed1976

Small RNA species of the HeLa cell: metabolism and subcellular localization.

The small molecular weight RNAs of the HeLa cell have been located in specific subcellular fractions. SnA is located in the nucleolus and is partially bonded to nucleolar 28S RNA. SnD, the most abundant of the small nuclear RNAs, is partially released from the nucleus when the nuclear preparation is briefly warmed. SnF is released from the nuclei when chromatin is digested with the micrococcal nuclease and not when pancreatic DNAase is used. The remainder of the small nuclear species remain in the nucleus following the digestion of chromatin and are concluded to be elements of the "nuclear skeleton." SnK is found predominantly in the cytoplasm, but migrates quantitatively to the nuclear fraction in the presence of high levels of actinomycin D. ScL is totally cytoplasmic and is partially bound to cell membranes. It is the 7S RNA found in oncornavirus virions. All the small nuclear RNAs appear initially in the cytoplasmic fraction before fixation in the nucleus. Two short-lived cytoplasmic species behave kinetically as precursors to the stable nuclear RNAs.

Cell Fractionation↗
PubMed2025

The Salmonella pathogenicity island 1-encoded small RNA InvR mediates post-transcriptional feedback control of the activator HilA in Salmonella.

UNLABELLED: Salmonella Pathogenicity Island 1 (SPI1) encodes a Type-3 secretion system (T3SS) essential for Salmonella invasion of intestinal epithelial cells. Many environmental and regulatory signals control SPI1 gene expression, but in most cases, the molecular mechanisms remain unclear. Many regulatory signals control SPI1 at a post-transcriptional level, and we have identified a number of small RNAs (sRNAs) that control the SPI1 regulatory circuit. The transcriptional regulator HilA activates the expression of the genes encoding the SPI1 T3SS structural and primary effector proteins. Transcription of hilA is controlled by the AraC-like proteins HilD, HilC, and RtsA. The hilA mRNA 5' untranslated region (UTR) is ~350 nucleotides in length and binds the RNA chaperone Hfq, suggesting it is a likely target for sRNA-mediated regulation. We used rGRIL-seq (reverse global sRNA target identification by ligation and sequencing) to identify sRNAs that bind to the hilA 5' UTR. The rGRIL-seq data, along with genetic analyses, demonstrate the SPI1-encoded sRNA invasion gene-associated RNA (InvR) base pairs at a site overlapping the hilA ribosome binding site. HilD and HilC activate both invR and hilA. InvR, in turn, negatively regulates the translation of the hilA mRNA. Thus, the SPI1-encoded sRNA InvR acts as a negative feedback regulator of SPI1 expression. Our results suggest that InvR acts to fine-tune SPI1 expression and prevents overactivation of hilA expression, highlighting the complexity of sRNA regulatory inputs controlling SPI1 and Salmonella virulence. IMPORTANCE: Salmonella Typhimurium infections pose a significant public health concern, leading to illnesses that range from mild gastroenteritis to severe systemic infection. Infection requires a complex apparatus that the bacterium uses to invade the intestinal epithelium. Understanding how Salmonella regulates this system is essential for addressing these infections effectively. Here, we show that the small RNA (sRNA) InvR imposes a negative feedback regulation on the expression of the invasion system. This work underscores the role of sRNAs in Salmonella's complex regulatory network, offering new insights into how these molecules contribute to bacterial adaptation and pathogenesis.

Genomic Islands↗
PubMed2005

Small RNA asymmetry in RNAi: function in RISC assembly and gene regulation.

RNAi is a conserved gene-specific regulatory mechanism, which silences target gene expression transcriptionally and post-transcriptionally. The RNAi machinery converts the sequence specific information of a long double stranded RNAs (dsRNAs) into small 21-22 nt long dsRNAs (siRNAs, miRNAs) which assemble into an effector complex, the RNA induced silencing complex (RISC). RISC assembly is asymmetric; one strand of an siRNA or a miRNA preferentially incorporates into the RNA-protein complex. Here, I review the rules of the asymmetric RISC formation and discuss their possible regulatory function in several steps in RNAi.

Animals↗
PubMed2006

Ebbie: automated analysis and storage of small RNA cloning data using a dynamic web server.

BACKGROUND: DNA sequencing is used ubiquitously: from deciphering genomes to determining the primary sequence of small RNAs (smRNAs). The cloning of smRNAs is currently the most conventional method to determine the actual sequence of these important regulators of gene expression. Typical smRNA cloning projects involve the sequencing of hundreds to thousands of smRNA clones that are delimited at their 5' and 3' ends by fixed sequence regions. These primers result from the biochemical protocol used to isolate and convert the smRNA into clonable PCR products. Recently we completed a smRNA cloning project involving tobacco plants, where analysis was required for approximately 700 smRNA sequences. Finding no easily accessible research tool to enter and analyze smRNA sequences we developed Ebbie to assist us with our study. RESULTS: Ebbie is a semi-automated smRNA cloning data processing algorithm, which initially searches for any substring within a DNA sequencing text file, which is flanked by two constant strings. The substring, also termed smRNA or insert, is stored in a MySQL and BlastN database. These inserts are then compared using BlastN to locally installed databases allowing the rapid comparison of the insert to both the growing smRNA database and to other static sequence databases. Our laboratory used Ebbie to analyze scores of DNA sequencing data originating from an smRNA cloning project. Through its built-in instant analysis of all inserts using BlastN, we were able to quickly identify 33 groups of smRNAs from approximately 700 database entries. This clustering allowed the easy identification of novel and highly expressed clusters of smRNAs. Ebbie is available under GNU GPL and currently implemented on http://bioinformatics.org/ebbie/. CONCLUSION: Ebbie was designed for medium sized smRNA cloning projects with about 1,000 database entries. Ebbie can be used for any type of sequence analysis where two constant primer regions flank a sequence of interest. The reliable storage of inserts, and their annotation in a MySQL database, BlastN comparison of new inserts to dynamic and static databases make it a powerful new tool in any laboratory using DNA sequencing. Ebbie also prevents manual mistakes during the excision process and speeds up annotation and data-entry. Once the server is installed locally, its access can be restricted to protect sensitive new DNA sequencing data. Ebbie was primarily designed for smRNA cloning projects, but can be applied to a variety of RNA and DNA cloning projects.

Algorithms↗
PubMed1995

Induction of a herpesvirus saimiri small RNA AU binding factor (AUBF70) activity and lymphokine mRNAs by T cell mitogens.

Herpesvirus saimiri (H. saimiri) can transform T lymphocytes and cause lymphoid tumors in rabbits and New World monkeys. H. saimiri-immortalized T cells express IL-2 and IL-4. The putative oncogenes of a group C strain of H. saimiri have been mapped to a region of the unique L-DNA which includes genes encoding four U-like small nuclear RNAs (HSUR1-HSUR4). Jurkat T cells express a 70 kD RNA binding factor (AUBF70) which binds HSUR2. Here we examined AUBF70 expression in resting and mitogen-stimulated human peripheral blood T cells and its sequence specificity and subcellular distribution. Band-shift assays demonstrated that resting human T cells express low amounts of AUBF70 which is induced by mitogen treatment. IL-2 and IL-4 mRNAs were co-induced with AUBF70 suggesting that AUBF70 is a positive regulator of lymphokine gene expression. Normal resting, mitogen-stimulated, and leukemic Jurkat T cells all express AUBF70 with virtually identical V8 proteolytic enzyme digestion patterns. Northern blots demonstrated that HSUR1 and HSUR2 are localized both in the nucleus and cytoplasm. HSUR2 accumulate in the cytoplasm in the presence of actinomycin D, which is consistent with re-transport of HSURs to the nucleus by (an) unstable factor(s). We hypothesize that HSUR1 and 2 transport AUBF70 from the cytoplasm to the nucleus; in the nucleus, AUBF70 binds and stabilizes lymphokine transcripts. Increased stability of lymphokine mRNAs could contribute to oncogenic transformation induced by H. saimiri.

Base Sequence↗
PubMed2004

Imprinted small RNA genes.

Genomic imprinting is an epigenetic phenomenon that results in differential expression of both alleles, depending on their parent of origin. We have recently identified many imprinted small non-coding RNA genes belonging to the C/D RNA and microRNA gene families, both of which are usually known to play key roles in post-transcriptional metabolism of specific genes (e.g. C/D RNAs guide ribose methylation of target RNAs while microRNAs elicit either translational repression or RNA interference). Although the functional and evolutionary significance of this association between C/D RNA genes, microRNA genes and genomic imprinting is still highly elusive, these observations provide a framework for further analysis of the potential role of small non-coding RNAs in epigenetic control.

Animals↗
PubMed2001

The Small RNA gene activator protein, SphI postoctamer homology-binding factor/selenocysteine tRNA gene transcription activating factor, stimulates transcription of the human interferon regulatory factor-3 gene.

Many small nuclear RNA gene promoters are activated by SphI postoctamer homology (SPH)-binding factor/selenocysteine tRNA gene transcription activating factor (SBF/Staf). Whereas this transcription factor was initially identified by its ability to bind to SPH elements in such promoters, it was more recently shown to have the capacity to activate transcription of a synthetic mRNA gene promoter through a distinct activation domain. Here, we show that the human interferon regulatory factor-3 (IRF-3) gene promoter contains a functional SPH element that is bound by SBF/Staf in vitro and in transfected cells.

Amino Acid Motifs↗
PubMed2004

3'-End polishing of the kinetoplastid spliced leader RNA is performed by SNIP, a 3'-->5' exonuclease with a Motley assortment of small RNA substrates.

In all trypanosomatids, trans splicing of the spliced leader (SL) RNA is a required step in the maturation of all nucleus-derived mRNAs. The SL RNA is transcribed with an oligo-U 3' extension that is removed prior to trans splicing. Here we report the identification and characterization of a nonexosomal, 3'-->5' exonuclease required for SL RNA 3'-end formation in Trypanosoma brucei. We named this enzyme SNIP (for snRNA incomplete 3' processing). The central 158-amino-acid domain of SNIP is related to the exonuclease III (ExoIII) domain of the 3'-->5' proofreading epsilon subunit of Escherichia coli DNA polymerase III holoenzyme. SNIP had a preference for oligo(U) 3' extensions in vitro. RNA interference-mediated knockdown of SNIP resulted in a growth defect and correlated with the accumulation of one- to two- nucleotide 3' extensions of SL RNA, U2 and U4 snRNAs, a five-nucleotide extension of 5S rRNA, and the destabilization of U3 snoRNA and U2 snRNA. SNIP-green fluorescent protein localized to the nucleoplasm, and substrate SL RNA derived from SNIP knockdown cells showed wild-type cap 4 modification, indicating that SNIP acts on SL RNA after cytosolic trafficking. Since the primary SL RNA transcript was not the accumulating species in SNIP knockdown cells, SL RNA 3'-end formation is a multistep process in which SNIP provides the ultimate 3'-end polishing. We speculate that SNIP is part of an organized nucleoplasmic machinery responsible for processing of SL RNA.

Animals↗
PubMed2002

Regulation and mode of action of the second small RNA activator of RpoS translation, RprA.

Translation of the stationary phase sigma factor RpoS is stimulated by at least two small RNAs, DsrA and RprA. DsrA disrupts an inhibitory secondary structure in the rpoS leader mRNA by pairing with the upstream RNA. Mutations in rprA and compensating mutations in the rpoS leader demonstrate that RprA interacts with the same region of the RpoS leader as DsrA. This is the first example of two different small RNAs regulating a common target. Regulation of these RNAs differs. DsrA synthesis is increased at low temperature. We find that RprA synthesis is regulated by the RcsC/RcsB phosphorelay system, previously found to regulate capsule synthesis and promoters of ftsZ and osmC. An rcsB null mutation abolishes the basal level, whereas mutations in rcsC that activate capsule synthesis also activate expression of the rprA promoter. An essential site with similarity to other RcsB-regulated promoters was defined in the rprA promoter. Activation of the RcsC/RcsB system leads to increased RpoS synthesis, in an RprA-dependent fashion. This work suggests a new signal for RpoS translation and extends the global regulation effected by the RcsC/RcsB system to coregulation of RpoS with capsule and FtsZ.

Bacterial Proteins↗