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PubMed2005

Identification and characterization of small RNAs involved in RNA silencing.

Double-stranded RNA (dsRNA) is a potent trigger of sequence-specific gene silencing mechanisms known as RNA silencing or RNA interference. The recognition of the target sequences is mediated by ribonucleoprotein complexes that contain 21- to 28-nucleotide (nt) guide RNAs derived from processing of the trigger dsRNA. Here, we review the experimental and bioinformatic approaches that were used to identify and characterize these small RNAs isolated from cells and tissues. The identification and characterization of small RNAs and their expression patterns is important for elucidating gene regulatory networks.

Animals↗
PubMed2006

Sequence-specific interference by small RNAs derived from adenovirus VAI RNA.

A virus-associated RNA (VAI) of adenoviruses is a cytoplasmic non-coding RNA and it plays an important role for viral replication in infected cells. VAI RNA transcripts, produced by RNA polymerase III (pol III), form tightly structured stems, which confer resistance to cellular defense systems. We demonstrate here that small RNAs of approximately 22 nucleotides are produced from a terminal stem region but not from an apical stem of VAI RNA. We determined the processing sites of VAI RNA by S1 nuclease mapping and further confirmed that the processed small RNA can act as small interfering RNAs (siRNAs) or as microRNAs (miRNAs) in transient transfection assays and during viral infection. Our data demonstrate that non-coding RNAs synthesized by pol III can be substrates for Dicer, and diced small RNAs might regulate cellular phenomena as siRNAs and miRNAs.

Adenoviridae↗
PubMed2006

Modulating the outer membrane with small RNAs.

MicF, one of the first chromosomally encoded regulatory small RNAs (sRNAs) to be discovered, was found to modulate the expression of OmpF, an abundant outer membrane protein. Several recent papers have now shown that this is not an isolated case. At least five other sRNAs also regulate the synthesis of outer membrane porins, and additional sRNAs modulate the expression of other outer membrane proteins. Here we review what is known about these sRNAs and discuss the implications of this regulation.

Bacterial Outer Membrane Proteins↗
PubMed1990

Characterization of U6 small nuclear RNA cap-specific antibodies. Identification of gamma-monomethyl-GTP cap structure in 7SK and several other human small RNAs.

The cap structure in human U6 small nuclear (sn)RNA, gamma-monomethylguanosine triphosphate (meGTP), was conjugated to human serum albumin and used as antigen to raise polyclonal antibodies in rabbits. The resulting antibodies reacted specifically with meGTP but not with GTP, GDP, GMP, meGMP, meATP, meCTP, meUTP, or with methyl phosphate in enzyme-linked immunosorbent assay and/or in radioimmunoassays. Although less efficiently, meGDP was also recognized by these antibodies. Indirect immunofluorescence studies with anti-meGTP antibodies showed predominantly nuclear immunofluorescence. Anti-meGTP antibodies immunoprecipitated intact U6 snRNA from a mixture of HeLa cell RNAs. In addition to the U6 snRNA, anti-meGTP antibodies immunoprecipitated several additional small RNAs that varied in length from approximately 50 to 330 nucleotides. These RNAs contained the meGTP cap structure and are structurally distinct from U6 snRNA. One of these meGTP-containing RNAs was found to be previously characterized 7SK RNA; human 7SK RNA synthesized in vitro also contained the same cap structure. Results obtained in this study provide evidence for the presence of gamma-monomethyl-GTP cap structure in a wide spectrum of human cellular RNAs. These antibodies will be useful in studying the structure and function of this new family of small RNAs.

Antibodies↗
PubMed1991

Short leader sequences may be transferred from small RNAs to pre-mature mRNAs by trans-splicing in Euglena.

Very closely related short sequences are present at the 5' end of cytoplasmic mRNAs in Euglena as evidenced by comparison of cDNA sequences and hybrid-arrested translation experiments. By cloning Euglena gracilis nuclear DNA and isolating the rbcS gene (encoding the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase), we have shown that the short leader sequence does not flank the nuclear gene sequence. The leader sequences were found to constitute the 5' extremities of a family of small RNAs. Sequencing six members of this family revealed a striking similarity to vertebrate U snRNAs. We propose that a trans-splicing mechanism transfers the spliced leader (SL) sequence from these small RNAs (SL RNAs) to pre-mature mRNAs. Transfer of leader sequences to mRNAs by trans-splicing has been shown only in trypanosomes where cis-splicing is unknown, and in nematodes where not more than 10% of the mRNAs have leader sequences. Our results strongly suggest that Euglena is a unique organism in which both a widespread trans-splicing and a cis-splicing mechanism co-exist.

Animals↗
PubMed2002

Analysis of a piwi-related gene implicates small RNAs in genome rearrangement in tetrahymena.

During development of the somatic macronucleus from the germline micronucleus in ciliates, chromosome rearrangements occur in which specific regions of DNA are eliminated and flanking regions are healed, either by religation or construction of telomeres. We identified a gene, TWI1, in Tetrahymena thermophila that is homologous to piwi and is required for DNA elimination. We also found that small RNAs were specifically expressed prior to chromosome rearrangement during conjugation. These RNAs were not observed in TWI1 knockout cells and required PDD1, another gene required for rearrangement, for expression. We propose that these small RNAs function to specify sequences to be eliminated by a mechanism similar to RNA-mediated gene silencing.

Animals↗
PubMed2005

Three small RNAs jointly ensure secondary metabolism and biocontrol in Pseudomonas fluorescens CHA0.

In many Gram-negative bacteria, the GacS/GacA two-component system positively controls the expression of extracellular products or storage compounds. In the plant-beneficial rhizosphere bacterium Pseudomonas fluorescens CHA0, the GacS/GacA system is essential for the production of antibiotic compounds and hence for biological control of root-pathogenic fungi. The small (119-nt) RNA RsmX discovered in this study, together with RsmY and RsmZ, forms a triad of GacA-dependent small RNAs, which sequester the RNA-binding proteins RsmA and RsmE and thereby antagonize translational repression exerted by these proteins in strain CHA0. This small RNA triad was found to be both necessary and sufficient for posttranscriptional derepression of biocontrol factors and for protection of cucumber from Pythium ultimum. The same three small RNAs also positively regulated swarming motility and the synthesis of a quorum-sensing signal, which is unrelated to N-acyl-homoserine lactones, and which autoinduces the Gac/Rsm cascade. Expression of RsmX and RsmY increased in parallel throughout cell growth, whereas RsmZ was produced during the late growth phase. This differential expression is assumed to facilitate fine tuning of GacS/A-controlled cell population density-dependent regulation in P. fluorescens.

Bacterial Proteins↗
PubMed1994

In vitro selection of small RNAs that bind to Escherichia coli phenylalanyl-tRNA synthetase.

Small RNAs were selected from a highly degenerate library on the basis of their ability to bind tightly to Escherichia coli phenylalanyl-tRNA synthetase (FRS). The 63 nucleotide library consisted of the acceptor stem and portions of the D and T stems of E. coli tRNA(Phe) flanking a 32 nucleotide randomized region. Because FRS binding relies on a correctly folded tRNA substrate, the selected variants from this library were expected to resemble tRNA(Phe) structure. After seven cycles of selection, the RNA library bound to FRS with similar affinity to that of the E. coli tRNA(Phe), but did not show detectable aminoacylation. Fourteen FRS-specific isolates were sequenced and found to contain an anticodon stem-loop including the anticodon triplet of tRNA(Phe). The tight-binding RNAs fell into two classes depending on the location of this step-loop within the sequence. The acceptor stem defined by the non-randomized sequence was also found to be essential for binding. Mutation of two residues within a common hexanucleotide sequence present in one of the classes reduced binding to FRS. Taken together, these results suggest that in order to bind RNAs tightly, FRS requires the simultaneous interaction of the anticodon stem-loop and acceptor stem, and additional sequences needed for proper folding. This approach should assist in the detection of motifs that resemble tRNA, but are too dissimilar to be identified by sequence comparison.

Base Sequence↗
PubMed2005

The expanding world of small RNAs in the hyperthermophilic archaeon Sulfolobus solfataricus.

Archaeal L7Ae is a multifunctional protein that binds to a distinctive K-turn motif in RNA and is found as a component in the large subunit of the ribosome, and in ribose methylation and pseudouridylation guide RNP particles. A collection of L7Ae-associated small RNAs were isolated from Sulfolobus solfataricus cell extracts and used to construct a cDNA library; 45 distinct cDNA sequences were characterized and divided into six groups. Group 1 contained six RNAs that exhibited the features characteristic of the canonical C/D box archaeal sRNAs, two RNAs that were atypical C/D box sRNAs and one RNA representative of archaeal H/ACA sRNA family. Group 2 contained 13 sense strand RNA sequences that were encoded either within, or overlapping annotated open reading frames (ORFs). Group 3 contained three sequences form intergenic regions. Group 4 contained antisense sequences from within or overlapping sense strand ORFs or antisense sequences to C/D box sRNAs. More than two-thirds of these sequences possessed K-turn motifs. Group 5 contained two sequences corresponding to internal regions of 7S RNA. Group 6 consisted of 11 sequences that were fragments from the 5' or 3' ends of 16S and 23S ribosomal RNA and from seven different tRNAs. Our data suggest that S. solfataricus contains a plethora of small RNAs. Most of these are bound directly by the L7Ae protein; the others may well be part of larger, transiently stable RNP complexes that contain the L7Ae protein as core component.

Base Sequence↗
PubMed1983

Transfer of the Epstein-Barr virus genes coding for small RNAs to human lymphoid cells with a vector carrying a dominant selectable marker.

Epstein-Barr virus (EBV)-negative, Burkitt-like lymphoma-derived cells were transformed with a transducing vector (pSV2-gpt) containing the Escherichia coli gene coding for xanthine-guanine phosphoribosyltransferase (XGPRT) and with a derivative of PSV2-gpt that carries the genes for the EBV-associated small RNAs on the EcoRI J fragment of B95-8 EBV DNA inserted at the unique EcoRI site (pJ-gpt). Cells transformed with PSV2-gpt and pJ-gpt express the E. coli gpt gene to approximately the same extent, judged by determinations of the XGPRT activity of cell extracts. Blot hybridisation experiments with restriction endonuclease-cleaved DNA from the transformants have revealed the presence of vector DNA sequences in the cells, at least some of which are most probably integrated into high mol. wt. chromosomal DNA. Northern blot hybridisation analysis of cytoplasmic RNA from pJ-gpt-transformed cells revealed the presence of an EcoRI J DNA complementary RNA species of the same size as the EBV DNA-encoded small RNAs found in EBV-transformed cells.

Burkitt Lymphoma↗
PubMed1981

Inter-RNA homology and possible roles of small RNAs.

The nucleotide sequence of a segment of U1 and U3b small RNAs (sRNAs) is shown to have a high complementarity with the nucleotide sequence of a part of the leader region of almost all eukaryotic genes studied so far. The complementary region of U3b is located in the unpaired segment of the secondary structure of U3b constructed by Reddy et al. (1979). A similar complementarity is also observed between these RNAs and the leader regions of eukaryotic viruses, but the complementary region is not always identical with that for eukaryotic genes. Complementarity is also observed between the 3' end of 18S rRNA and a segment of U1 or U3b which is almost contiguous to the region complementary with mRNA. These observations suggest that U1 and U3b may be involved in mRNA processing and transport in the nucleus or in translation in the cytoplasm. In addition to U1 and U3b, another sRNA, i.e., 4.5S RNAI, is shown to have segments which are homologous to the Hogness box of the flanking region of gene and the Proudfoot-Brownlee (PB) box of mRNA near the poly(A) attachment site. The two segments which are complementary with these boxes are located almost contiguously on a co-joined loop of the secondary structure of 4.5S RNAI constructed by Ro-Choi et al. (1972). Since the Hogness box and PB box are both considered as a recognition site by the RNA polymerase, it is possible that 4.5S RNAI is involved in mediating gene transcription.

Animals↗
PubMed2005

Small RNAs in human brain development and disorders.

Small RNA is a variable and abundant type of non-coding RNAs in brain. The function of these RNAs is mainly unknown. A specific class of small RNA, microRNA, is dynamically regulated in neurogenesis and in embryo brain development. The genes for synaptic formation and some mental retardation disorders are putative targets for microRNA predicted by computational algorithms. The molecular pathways for mental development, common forms of autisms, schizophrenia, and affective disorders have yet to be elucidated. The hypothesis proposed here is that small regulatory RNAs, specifically microRNAs, play a role in human brain development and pathogenesis of brain disorders, especially of neurodevelopmental conditions. Pilot tests using comprehensive arrays of microRNAs demonstrate that microRNAs derived from postmortem human brains are applicable for microRNA expression profiling. The abundant expression of many regulatory small RNAs in human brain implies their biological role that must be tested by functional assays in neurons and by genetic and comparative expression profiling.

Brain↗
PubMed2001

An extensive class of small RNAs in Caenorhabditis elegans.

The lin-4 and let-7 antisense RNAs are temporal regulators that control the timing of developmental events in Caenorhabditis elegans by inhibiting translation of target mRNAs. let-7 RNA is conserved among bilaterian animals, suggesting that this class of small RNAs [microRNAs (miRNAs)] is evolutionarily ancient. Using bioinformatics and cDNA cloning, we found 15 new miRNA genes in C. elegans. Several of these genes express small transcripts that vary in abundance during C. elegans larval development, and three of them have apparent homologs in mammals and/or insects. Small noncoding RNAs of the miRNA class appear to be numerous and diverse.

Animals↗
PubMed1987

A library of trimethylguanosine-capped small RNAs in Physarum polycephalum.

We have constructed a cDNA library for the trimethylguanosine-capped small RNAs (sRNAs) in the acellular slime mold Physarum polycephalum. Capped sRNAs were purified from total cellular RNA of vegetative microplasmodia by preparative immunoprecipitation with anti-trimethylguanosine antibody. The purified RNA was analyzed by polyacrylamide gel electrophoresis. Approx. eleven different capped sRNAs were observed with a size range of 70-204 nucleotides (nt). Based on their approximate sizes, the presence of trimethylguanosine cap, and the presence of a lupus type-Sm antigen, molecules U1-U7 (excluding U3) were identified. Further confirmation of the identity of molecule U1a was established by Northern hybridization, U4a by colony hybridization, and U6 and U7a by direct chemical sequence analysis. Purified capped sRNAs were tailed with oligo(A), and inserted into oligo(dT)-tailed plasmid pCDV1. The cDNAs were used to transform Escherichia coli strain HB101. Approx. 1.9 X 10(5) ampicillin-resistant (ApR) transformants were obtained per microgram of tailed sRNA. Dot-blot hybridization, using Physarum RNA precipitated with anti-cap antibody as a probe, indicated that approx. 94% of the ApR colonies contained recombinant DNAs. The library was screened by colony hybridization using heterologous sRNA probes. Clones hybridizing with heterologous sRNAs U1, U2, U4 and U7 were each represented in the library in approximately the same frequency as their relative abundance in the Physarum sRNA population they were derived from. The insert of one Physarum U4 clone was sequenced and was found to have 57.1% homology with nt 1-91 of the published sequence for rat U4 RNA. A 12-nt 'functional' subdomain of the rat U4 molecule was 83.3% conserved in Physarum U4.

Autoantigens↗
PubMed1980

6.5 S RNA; preliminary characterisation of unusual small RNAs in Trypanosoma brucei.

In this paper we report the preliminary characterisation of some unusual small rRNAs isolated from Trypanosoma brucei. These molecules correspond closely in size and properties to the 5 S and 5.8 S rRNAs of other eukaryotes and in addition there are two further small RNAs which we designate 6.5 S RNA. These have been characterised by sucrose gradient centrifugation and electrophoresis in denaturing gels. The two 6.5 S RNAs may be distinguished from each other on the basis of their size and the different conditions required for their dissociation from the ribosomes. They are present in the same abundance as 5.8 S RNA suggesting that they occur once per ribosome.

Animals↗
PubMed1988

Isolation and characterization of the genes for two small RNAs of herpesvirus papio and their comparison with Epstein-Barr virus-encoded EBER RNAs.

Genes for the Epstein-Barr virus-encoded RNAs (EBERs), two low-molecular-weight RNAs encoded by the human gammaherpesvirus Epstein-Barr virus (EBV), hybridize to two small RNAs in a baboon cell line that contains a similar virus, herpesvirus papio (HVP). The genes for the HVP RNAs (HVP-1 and HVP-2) are located together in the small unique region at the left end of the viral genome and are transcribed by RNA polymerase III in a rightward direction, similar to the EBERs. There is significant similarity between EBER1 and HVP-1 RNA, except for an insert of 22 nucleotides which increases the length of HVP-1 RNA to 190 nucleotides. There is less similarity between the sequences of EBER2 and HVP-2 RNA, but both have a length of about 170 nucleotides. The predicted secondary structure of each HVP RNA is remarkably similar to that of the respective EBER, implying that the secondary structures are important for function. Upstream from the initiation sites of all four RNA genes are several highly conserved sequences which may function in the regulation of transcription. The HVP RNAs, together with the EBERs, are highly abundant in transformed cells and are efficiently bound by the cellular La protein.

Autoantigens↗
PubMed2002

Micro-RNAs: small is plentiful.

Two small temporally regulated RNAs (stRNAs)* of approximately 22 nucleotides regulate timing of gene expression during development of the nematode C. elegans. This regulation occurs at a posttranscriptional, presumably translational, level and is distinct from RNA interference (RNAi). One of the two stRNAs, let-7, as well as its target gene, lin-41, are highly conserved even in humans, suggesting a wide employment of stRNA-mediated gene regulation. Recent reports indicate that these two stRNAs are indeed likely to represent only the tip of an iceberg with hundreds or more of additional micro-RNAs (miRNAs) existing in metazoans. miRNAs might thus be previously underestimated key participants in the field of gene regulation.

Animals↗
PubMed2006

Running interference: prospects and obstacles to using small interfering RNAs as small molecule drugs.

RNA interference (RNAi) is a well-conserved, ubiquitous, endogenous mechanism that uses small noncoding RNAs to silence gene expression. The endogenous small RNAs, called microRNAs, are processed from hairpin precursors and regulate important genes involved in cell death, differentiation, and development. RNAi also protects the genome from invading genetic elements, encoded by transposons and viruses. When small double-stranded RNAs, called small interfering (si)RNAs, are introduced into cells, they bind to the endogenous RNAi machinery to disrupt the expression of mRNAs containing complementary sequences with high specificity. Any disease-causing gene and any cell type or tissue can potentially be targeted. This technique has been rapidly utilized for gene-function analysis and drug-target discovery and validation. Harnessing RNAi also holds great promise for therapy, although introducing siRNAs into cells in vivo remains an important obstacle. Pilot siRNA clinical studies began just three years after the discovery that RNAi works in mammalian cells. This review discusses recent progress and obstacles to using siRNAs as small molecule drugs.

Animals↗