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Cellular dynamics of small RNAs.

This review highlights the unexpectedly complicated nuclear egress and nuclear import of small RNAs. Although nucleus/cytoplasm trafficking was thought to be restricted to snRNAs of many, but not all, eukaryotes, recent data indicate that such traffic may be more common than previously appreciated. First, in conflict with numerous previous reports, new information indicates that Saccharomyces cerevisiae snRNAs may cycle between the nucleus and the cytoplasm. Second, recent studies also provide evidence that other small RNAs that function exclusively in the nucleus-the budding yeast telomerase RNA and possibly small nucleolar RNAs-may exit to the cytoplasm, only to return to the nucleus. Third, nucleus/cytoplasm cycling of RNAs also occurs for RNAs that function solely in the cytoplasm, as it has been discovered that cytoplasmic tRNAs of budding yeast travel "retrograde" to the nucleus and, perhaps, back again to the cytoplasm to function in protein synthesis. Fourth, there is at least one example in ciliates of small double-stranded RNAs traveling multiple cycles between the cytoplasm and distinct nuclei to direct genome structure. This report discusses data that support or argue against nucleus/cytoplasm bidirectional movement for each category of small RNA and the possible roles that such movement may serve.

Animals↗
PubMed1998

Adenylation of small RNAs in human cells. Development of a cell-free system for accurate adenylation on the 3'-end of human signal recognition particle RNA.

The 3'-end sequences of several human small RNAs were determined, and the results show that a fraction of human cytoplasmic 7SL, ribosomal 5S, and nuclear U2, U6, and 7SK small RNAs contain a post-transcriptionally added adenylic acid residue on their 3'-ends. Incubation of HeLa cell extract in vitro in the presence of [alpha-32P]ATP resulted in labeling of several small RNAs including ribosomal 5S and cytoplasmic 7SL as well as U2 and U6 small nuclear RNAs. Analysis of 7SL RNA labeled in this in vitro adenylation system showed that a single adenylic acid residue is added to the 3'-end. These results show that the adenylation observed in the in vitro system reflects the post-transcriptional adenylation occurring in vivo.

Adenosine Triphosphate↗
PubMed2002

Methylphosphate cap structure in small RNAs reduces the affinity of RNAs to La protein.

La protein is an abundant 47-kDa phosphoprotein found mostly in the nucleus of eukaryotic cells with a small fraction present in the cytoplasm. Nascent RNA transcripts synthesized by RNA polymerase III are known to be associated with La protein. This binding has been shown to occur to the 3' end of RNA via RNA recognition motifs and to the 5' triphosphate via the Walker A motif of the La protein. In this study, we developed an in vitro immunoprecipitation assay to quantitate the 5' ppp-dependent binding of small RNAs to the human La protein. Using this assay, we found that oligonucleotides five bases or longer bind to the human La protein in a 5' ppp-dependent manner, pppG did not bind to La protein in this assay. In addition, CH3pppN cap structure present on the 5' ends of U6 and B2 small RNAs reduced the ability of these RNAs to bind the human La protein. These data show that Walker motif in the human La protein can bind to short RNAs containing 5' ppp and removal of 5' ppp from RNAs, or modification of 5' pppN to CH3pppN or m7GpppN, significantly reduces the ability of small RNAs to bind the human La protein. These data suggest that one of the functions of methylphosphate cap structure in U6 snRNA and B2 RNAs is possibly to reduce the affinity of these RNAs to La protein.

Amino Acid Motifs↗
PubMed2006

Arabidopsis endogenous small RNAs: highways and byways.

Several classes of endogenous small RNAs of 20-26 nucleotides, such as microRNAs and small interfering (si)RNAs, have been described in plants and animals. Arabidopsis currently seems to be an exception because a novel genetically distinguishable small RNA class, termed natural-antisense transcript derived siRNAs, was recently identified. Pathways for the biogenesis and function of each class are already well characterized, enabling their classification on the basis of both size and proteins required for their function. However, each pathway requires proteins belonging to conserved protein families, and recent genetic studies confirmed an expected partial redundancy between members of the Dicer-like (DCL) family. Moreover, several experimental results indicate redundancies among double-strand RNA binding proteins and ARGONAUTE proteins, which probably create a complex network of small RNA pathways.

Arabidopsis↗
PubMed2005

Optimizing splinted ligation of highly structured small RNAs.

The synthesis of highly structured small RNAs containing nonstandard nucleotides is of high interest for structural and functional investigations. A general approach is the joining, by T4 DNA ligase-mediated splinted ligation, of two or more RNA fragments, each of which may contain its own set of modified nucleotides. The RNA fragments hybridize with a complementary DNA splint to form a ternary ligation-competent-complex (LCC), which is then turned over by the DNA ligase. We studied the formation of the LCC and its precursors using size exclusion chromatography combined with a fluorescence detector. The spatial proximity of two cyanine-dye-labeled RNA fragments in LCCs was detected by monitoring FRET. An observed correlation of LCC formation and ligation yields suggests the use of long splints to stabilize LCCs. Splint oligos of increasing length, which in general appear to reduce the number of different hybridization intermediate species found in a reaction mixture, were applied to the synthesis by T4-DNA-ligation of two highly structured target molecules, one a 73 mer tRNA, the other a 49 mer synthetic ribozyme. A stable LCC could be isolated and turned over with>95% ligation efficiency. In conclusion, the use of long splints presents a generally applicable means to overcome the low propensity of highly structured RNAs for hybridization, and thus to significantly improve ligation efficiencies.

Base Sequence↗
PubMed2000

Effect of 3' terminal adenylic acid residue on the uridylation of human small RNAs in vitro and in frog oocytes.

It is known that several small RNAs including human and Xenopus signal recognition particle (SRP) RNA, U2 small nuclear RNA (snRNA) and 7SK RNAs are posttranscriptionally adenylated, whereas U6 snRNA and ribosomal 5S RNA are posttranscriptionally uridylated on their 3' ends. In this study, we provide evidence that a small fraction of U6 snRNA and 5S ribosomal RNA molecules from human as well as Xenopus oocytes contain a single posttranscriptionally added adenylic acid residue on their 3' ends. These data show that U6 snRNA and 5S rRNAs are posttranscriptionally modified on their 3' ends by both uridylation and adenylation. Although the SRP RNA, 7SK RNA, 5S RNA, and U6 snRNA with the uridylic acid residue on their 3' ends were readily uridylated, all these RNAs with posttranscriptionally added adenylic acid residue on their 3' ends were not uridylated in vitro, or when U6 snRNA with 3' A(OH) was injected into Xenopus oocytes. These results show that the presence of a single posttranscriptionally added adenylic acid residue on the 3' end of SRP RNA, U6 snRNA, 5S rRNA, or 7SK RNA prevents 3' uridylation. These data also show that adenylation and uridylation are two competing processes that add nucleotides on the 3' end of some small RNAs and suggest that one of the functions of the 3' adenylation may be to negatively affect the 3' uridylation of small RNAs.

Adenosine Monophosphate↗
PubMed2007

A structured viroid RNA serves as a substrate for dicer-like cleavage to produce biologically active small RNAs but is resistant to RNA-induced silencing complex-mediated degradation.

RNA silencing is a potent means of antiviral defense in plants and animals. A hallmark of this defense response is the production of 21- to 24-nucleotide viral small RNAs via mechanisms that remain to be fully understood. Many viruses encode suppressors of RNA silencing, and some viral RNAs function directly as silencing suppressors as counterdefense. The occurrence of viroid-specific small RNAs in infected plants suggests that viroids can trigger RNA silencing in a host, raising the question of how these noncoding and unencapsidated RNAs survive cellular RNA-silencing systems. We address this question by characterizing the production of small RNAs of Potato spindle tuber viroid (srPSTVds) and investigating how PSTVd responds to RNA silencing. Our molecular and biochemical studies provide evidence that srPSTVds were derived mostly from the secondary structure of viroid RNAs. Replication of PSTVd was resistant to RNA silencing, although the srPSTVds were biologically active in guiding RNA-induced silencing complex (RISC)-mediated cleavage, as shown with a sensor system. Further analyses showed that without possessing or triggering silencing suppressor activities, the PSTVd secondary structure played a critical role in resistance to RISC-mediated cleavage. These findings support the hypothesis that some infectious RNAs may have evolved specific secondary structures as an effective means to evade RNA silencing in addition to encoding silencing suppressor activities. Our results should have important implications in further studies on RNA-based mechanisms of host-pathogen interactions and the biological constraints that shape the evolution of infectious RNA structures.

Arabidopsis↗
PubMed2005

RNA-conjugated template-switching RT-PCR method for generating an Escherichia coli cDNA library for small RNAs.

The cDNA conversion of RNA molecules is a prerequisite for their analysis. In the case of prokaryotic RNAs, cDNA conversion is difficult due to a lack of the long poly(A) tails that are found in eukaryotic mRNAs. The full cDNAs for eukaryotic mRNAs can be amplified by the reverse transcription polymerase chain reaction (RT-PCR) using the template-switching method together with an oligo(dT) primer. To amplify the full cDNAs for prokaryotic RNAs, we modified the template-switching RT-PCR method by adopting an RNA linker at the 3' end of the target RNAs. Using this method, which we named as RNA-conjugated template-switching RT-PCR (RC/TS RT-PCR), we constructed a cDNA library for small RNAs from cold-shock-treated Escherichia coli cells. To confirm that the cDNAs were amplified by RC/TS RT-PCR without a loss of sequence information, clones carrying the 6S RNA sequence were analyzed from the cDNA library for small RNAs ranging from 130 to 350 nt. We found that the 6S RNA sequences were fully converted into the corresponding cDNAs, confirming that RC/TS RT-PCR is a useful method for constructing a cDNA library for small RNAs in E. coli. This method can be also used to construct a cDNA library for non-poly(A)-containing RNAs from eukaryotic cells.

Blotting, Northern↗
PubMed2004

World of small RNAs: from ribozymes to siRNA and miRNA.

RNAs, besides bridging genetic information to proteins, the major determinants of bio-structures and functions, serve as active regulators of gene expression. Initiated nearly 20 years ago with ribozymes (the small RNAs with catalytic activity providing fine tuning of gene expression and function, used as molecular scissors and tools for gene discovery), an era of more complex and coordinated gene regulation by small RNAs, siRNA, and miRNA has recently started. Simple nucleotide complementarity results in highly ordered and regulated events, such as assembly of RNA and proteins, resulting in gene silencing either by mRNA degradation or suppression of translation. This article reviews our contributions to the understanding of structure, the function of small RNAs, their use in biotechnology, and the understanding of phenotypes such as apoptosis, metastasis, and differentiation.

Animals↗
PubMed2001

HC-Pro suppression of transgene silencing eliminates the small RNAs but not transgene methylation or the mobile signal.

Post-transcriptional gene silencing (PTGS) is a sequence-specific RNA degradation mechanism that is widespread in eukaryotic organisms. It is often associated with methylation of the transcribed region of the silenced gene and with accumulation of small RNAs (21 to 25 nucleotides) homologous to the silenced gene. In plants, PTGS can be triggered locally and then spread throughout the organism via a mobile signal that can cross a graft junction. Previously, we showed that the helper component-proteinase (HC-Pro) of plant potyviruses suppresses PTGS. Here, we report that plants in which PTGS has been suppressed by HC-Pro fail to accumulate the small RNAs associated with silencing. However, the transgene locus of these plants remains methylated. Grafting experiments indicate that HC-Pro prevents the plant from responding to the mobile silencing signal but does not eliminate its ability to produce or send the signal. These results demonstrate that HC-Pro functions downstream of transgene methylation and the mobile signal at a step preceding accumulation of the small RNAs.

Algorithms↗
PubMed1997

Probing RNA-protein interactions using pyrene-labeled oligodeoxynucleotides: Qbeta replicase efficiently binds small RNAs by recognizing pyrimidine residues.

Binding of small RNAs by the RNA-dependent RNA polymerase of coliphage Qbeta was studied utilizing a fluorometric assay. A DNA oligonucleotide probe of sequence 5'-d(TTTTTCC) was 5'-end-labeled with pyrene. In this construct, the proximal thymine residues efficiently quench the fluorophore emission in solution. Upon stoichiometric binding of one probe per polymerase molecule, the pyrene steady-state fluorescence increases by two orders of magnitude, the fluorescence anisotropy increases, and a long fluorescence lifetime component of 140 ns appears. With addition of replicable RNA, steady-state fluorescence decreases in a concentration dependent manner and the long lifetime component is lost. This observation most likely reflects displacement of the pyrene-labeled probe from the proposed nucleic acid binding site II of Qbeta replicase. The effect was utilized to access binding affinities of different RNAs to this site in a reverse titration assay format. In 10 mM sodium phosphate (pH 7.0), 100 mM NaCl, at 16 degrees C, equilibrium dissociation constants for different template midi- and minivariant RNAs were calculated to be in the nanomolar range. In general, the minus and plus strands, concomitantly synthesized by Qbeta replicase during replication, exhibited discriminative affinities, while their hybrid bound less efficiently than either of the single strands. Different non-replicable tRNAs also bound to the polymerase with comparable dissociation constants. By titration with DNA homo-oligonucleotides it was shown that the probed site on Qbeta replicase does not require a 2' hydroxyl group for binding nucleic acids, but recognizes pyrimidine residues. Its interaction with thymine is lost in an A.T base-pair, while that with cytosine is retained after Watson-Crick base-pairing. These findings can explain the affinities of RNA-Qbeta replicase interactions reported here and in earlier investigations. The sensitivity of the described fluorometric assay allows detection of RNA amplification by Qbeta replicase in real-time.

Base Sequence↗
PubMed2006

Brain-specific small nucleolar RNAs.

Small nucleolar RNAs (snoRNAs) are a group of noncoding RNAs that function mainly as guides for modification of ribosomal RNAs (rRNAs) and small nuclear RNAs (snRNAs). A subgroup of snoRNAs was found to be predominantly expressed in the brain; and interestingly, these brain-specific snoRNAs (b-snoRNAs) appear not to be involved in modification of rRNAs and snRNAs, raising the question of what their function and targets might be. Expression studies of b-snoRNAs in mice have shown potential involvement of two b-snoRNAs, MBII-48 and MBII-52, in learning and memory. HBII-52, the human homolog of MBII-52, appears to be involved with regulation of 5-HT(2C) receptor subunit mRNA. Furthermore, several reports link the disruption of expression of a specific b-snoRNA, HBII-85, with a neurobehavioral disorder, Prader-Willi syndrome. This paper reviews the current knowledge of the properties, expression, and functions of b-snoRNAs.

Animals↗
PubMed1983

Small RNAs from Drosophila KC-H cells. Characterization of nuclear and cytoplasmic 7S subspecies.

The metabolic characteristics and nuclear and cytoplasmic localization of Drosophila 7 S small RNAs were investigated. It was found that although most Drosophila small RNAs show electrophoretic mobilities similar to those of mammalian cells, distinct metabolic behaviour can be detected in three 7 S subspecies (d1, d3 and d4) which were found not to be class III RNAs as reported for mammalian 7 S RNAs. Drosophila d3 and d4 were found to be localized in the nucleus and to be very rapidly labelled minor subspecies turning over with a half-life of less than 6 h. Although 7 S-d1 migrates close to mammalian 7 S-K, it is a major subspecies in Drosophila, is localized mostly in the nucleus and turns over with a half-life of less than 10 h in contrast with mammalian 7 S-K which has been observed to be quite stable.

Animals↗
PubMed1988

Small RNAs of the silkworm, Bombyx mori as revealed by in vitro capping and in vitro transcription.

1. We characterized several small RNAs in various tissues and strains of the silkworm, Bombyx mori by the in vitro capping and in vitro transcription. 2. Three subspecies of 7SL RNA, which is believed to be involved in protein secretion, were detected in the commercial strains. Structural differences among the three molecules were due to the nucleotide substitution at two positions or more. 3. A very AU-rich RNA, which is about 500 bases long, was always present in any tissue of Bombyx mori while 55 base RNA was only observed in a substantial amount in the fat body and culture cells. 4. These small RNAs were also observed among the in vitro transcripts of total Bombyx DNA in the cell-free extracts from the silkgland and fat body.

Animals↗
PubMed2006

Remodelling of the Escherichia coli outer membrane by two small regulatory RNAs.

Small non-coding RNAs that play important regulatory roles exist in numerous organisms. In Escherichia coli, about 60 small RNAs have been found and those that have been studied are involved in the response and adaptation to different stresses. RygA and RygB, two of these small RNAs, were identified on the basis of their conservation between different species and their ability to bind Hfq. They are adjacent on the chromosome and have sequence similarity at their 5' and 3' ends but distinct central regions, suggesting that they could regulate the expression of both common and distinct genes. A screen using a multicopy E. coli library led to identification of the response regulator OmpR and its associated sensor kinase EnvZ as positive regulators of rygA and rygB transcription. Therefore, RygA and RygB were renamed OmrA and OmrB respectively (for OmpR-regulated sRNAs A and B). When expressed at high levels, OmrA and OmrB RNAs negatively regulate the expression of several genes encoding multiple outer membrane proteins, including cirA, fecA, fepA and ompT. Taken together, these data suggest that OmrA and OmrB participate in the regulation of outer membrane composition in response to environmental conditions.

Bacterial Outer Membrane Proteins↗
PubMed2004

The 3' termini of small RNAs in Trypanosoma brucei.

Post-transcriptional 3' end formation is an essential step in the maturation of most small RNAs. Knowledge of the precise 3' ends of mature RNAs is essential for defining 3'-processing activities. We have mapped the mature 3' ends of Spliced Leader RNA, the U1, U2, U4, U5, and U6 small nuclear RNAs, the U3 small nucleolar RNA, and the 5S and 5.8S ribosomal RNAs by ligation-mediated PCR in Trypanosoma brucei. With the exception of U5, two classes of 3' ends were observed: flush with the base of a stem-loop structure, and 1-2 nt extended from a stem-loop. Multiple mechanisms and structural features are likely to influence 3' end maturation of RNAs.

Animals↗
PubMed1999

Small RNAs in Escherichia coli.

Bacterial cells contain several small RNAs (sRNAs) that are not translated. These stable, abundant RNAs act by multiple mechanisms, such as RNA-RNA basepairing, RNA-protein interactions and intrinsic RNA activity, and regulate diverse cellular functions, including RNA processing, mRNA stability, translation, protein stability and secretion.

Base Sequence↗
PubMed2005

Extensive 3' modification of plant small RNAs is modulated by helper component-proteinase expression.

RNA silencing is an evolutionarily conserved process in eukaryotes that represses gene expression by using 21- to 24-nt guide RNAs to mediate mRNA cleavage or translational inhibition. Plants have two distinct groups of silencing-associated small RNAs (smRNAs): the micro RNAs (miRNAs) and the small interfering RNAs (siRNAs). A recent report by Yu et al. [Yu, B., Yang, Z., Li, J., Minakhina, S., Yang, M., Padgett, R. W., Steward, R. & Chen, X. (2005) Science 307, 932-935] has shown that plant miRNAs are modified at their 3' termini with a methyl group. Here, we show that a large fraction of all silencing-associated smRNAs in tobacco are modified; this modification occurs on the 2' hydroxyl of the terminal ribose and significantly reduces the cloning efficiency of these modified smRNAs. Expression of the strong silencing suppressor P1/helper-component proteinase results in a marked decrease in the 3'-terminal modification of viral siRNAs but does not significantly affect the modification of endogenous miRNAs and 24-nt siRNAs. The differential modification mediated by helper-component proteinase expression implies that exogenous and endogenous smRNAs are processed through independent pathways that are isolated by subcellular compartmentalization and/or the association with distinct Dicer complexes. The degree of terminal modification may play an important role in regulating the extent to which primary smRNA signals can be amplified by RNA-dependent RNA polymerases.

Gene Expression Regulation, Plant↗