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PubMed2002

RNAi in human cells: basic structural and functional features of small interfering RNA.

We investigated the mechanism of RNA interference (RNAi) in human cells. Here we demonstrate that the status of the 5' hydroxyl terminus of the antisense strand of a siRNA determines RNAi activity, while a 3' terminus block is tolerated in vivo. 5' hydroxyl termini of antisense strands isolated from human cells were phosphorylated, and 3' end biotin groups were not efficiently removed. We found no requirement for a perfect A-form helix in siRNA for interference effects, but an A-form structure was required for antisense-target RNA duplexes. Strikingly, crosslinking of the siRNA duplex by psoralen did not completely block RNA interference, indicating that complete unwinding of the siRNA helix is not necessary for RNAi activity in vivo. These results suggest that RNA amplification by RNA-dependent RNA polymerase is not essential for RNAi in human cells.

Animals↗
PubMed1996

Identification of a region of Bacillus subtilis Ffh, a homologue of mammalian SRP54 protein, that is essential for binding to small cytoplasmic RNA.

Bacillus subtilis Ffh and scRNA are homologues of mammalian SRP54 and SRP RNA, respectively, which are components of the eukaryotic signal recognition particle (SRP). Ffh (446 amino acids) interacts with scRNA to form a stable complex in vivo. Here, we identified an RNA-binding domain of Ffh. The results obtained using a series of deletion mutants show that amino acid positions 364 to 432 in the C-terminal region of Ffh correlates with its ability to bind RNA. The amino acid sequence of this region is well conserved among members of the SRP54 protein family. This sequence contains two hydrophobic regions (h2, 364 to 391, and h3, 416 to 435), separated by the positively charged amino acid motif, 398RRKRIAKGSG407. Among the basic amino acid residues in this region, Arg-401 was essential for binding to scRNA, but Arg-399 and Lys-400 were not. The co-existence of Arg-398 and Lys-404 was necessary for the same affinity as wild type Ffh. The two glycine residues of the 405GSG407 were also essential. MH23 peptide (91 amino acids) encompassing from 356 to 446, consisting of h2-RRKRIAKGSG-h3, bound scRNA with the same affinity as wild type Ffh, whereas a 24-amino acid synthetic peptide 392DIINASRRKRIAKGSGTSVQEVNR415 did not. The region containing two hydrophobic segments separated by the positively charged motif is the minimal requirement of Ffh for RNA binding.

Amino Acid Sequence↗
PubMed1994

Mutations in an essential U2 small nuclear RNA structure cause cold-sensitive U2 small nuclear ribonucleoprotein function by favoring competing alternative U2 RNA structures.

Mutations in stem-loop IIa of yeast U2 RNA cause cold-sensitive growth and cold-sensitive U2 small nuclear ribonucleoprotein function in vitro. Cold-sensitive U2 small nuclear RNA adopts an alternative conformation that occludes the loop and disrupts the stem but does so at both restrictive and permissive temperatures. To determine whether alternative U2 RNA structure causes the defects, we tested second-site mutations in U2 predicted to disrupt the alternative conformation. We find that such mutations efficiently suppress the cold-sensitive phenotypes and partially restore correct U2 RNA folding. A genetic search for additional suppressors of cold sensitivity revealed two unexpected mutations in the base of an adjacent stem-loop. Direct probing of RNA structure in vivo indicates that the suppressors of cold sensitivity act to improve the stability of the essential stem relative to competing alternative structures by disrupting the alternative structures. We suggest that many of the numerous cold-sensitive mutations in a variety of RNAs and RNA-binding proteins could be a result of changes in the stability of a functional RNA conformation relative to a competing structure. The presence of an evolutionarily conserved U2 sequence positioned to form an alternative structure argues that this region of U2 is dynamic during the assembly or function of the U2 small nuclear ribonucleoprotein.

Base Sequence↗
PubMed1987

Human U3 small nucleolar RNA genes are localized to the nucleoplasm.

U3 RNA, an abundant, conserved, capped, small RNA localized to the nucleolar compartment of eukaryotic cells, its implicated in the processing of pre-ribosomal RNA. The genes for U1 and U2 snRNAs (small nuclear RNAs) are clustered and present in the nucleoplasmic DNA; however, the localization of U3 snRNA genes is not known. DNAs, isolated from HeLa cell nuclei and nucleoli, were hybridized with labeled probes corresponding to the 5'-flanking and the coding regions of the human U3 snRNA gene. The intensity of signals obtained with both the probes were 5-6 fold greater in nuclear DNA, compared to nucleolar DNA. With ribosomal gene probe, the nucleolar DNA had sixfold more intense signal than nuclear DNA. These results indicate that genes for U3 snRNA are in the nucleoplasm. Therefore, U3 snRNA, like 5S ribosomal RNA, appears to be synthesized in the nucleoplasm and transported to the nucleolus.

Cell Compartmentation↗
PubMed2005

Fully 2'-modified oligonucleotide duplexes with improved in vitro potency and stability compared to unmodified small interfering RNA.

We have identified a small interfering RNA (siRNA) motif, consisting entirely of 2'-O-methyl and 2'-fluoro nucleotides, that displays enhanced plasma stability and increased in vitro potency. At one site, this motif showed remarkable >500-fold improvement in potency over the unmodified siRNA. This marks the first report of such a potent fully modified motif, which may represent a useful design for therapeutic oligonucleotides.

Animals↗
PubMed1998

Molecular cloning of a cDNA encoding human SPH-binding factor, a conserved protein that binds to the enhancer-like region of the U6 small nuclear RNA gene promoter.

Many vertebrate small nuclear RNA gene promoters contain an SPH motif in their distal control regions that can confer transcriptional stimulation by RNA polymerase II or RNA polymerase III. Using the human U6 gene SPH motif as a probe, we isolated a cDNA encoding human SPH-binding factor (hSBF) from a HeLa cell expression library. The coding region of hSBF is almost identical to ZNF143, a 626 amino acid, seven zinc finger protein of previously unknown function. Furthermore, the predicted amino acid sequence of hSBF is highly homologous to Xenopus laevis and mouse Staf proteins, that bind to SPH motifs and stimulate transcription of selenocysteine tRNA gene promoters. Recombinant hSBF expressed in vitro or from Escherichia coli bound specifically to the human U6 gene SPH motif as shown by DNase I footprinting and electrophoretic mobility shift assays using various mutant SPH sites as competitors. Antibodies prepared against recombinant hSBF inhibited assembly of native SBF-DNA complexes. Immunodepleted HeLa S100 transcription extract no longer supported elevated levels of transcription by RNA polymerase III from a U6 promoter containing an SPH motif, whereas addition of recombinant hSBF protein to the immunodepleted extract reconstituted stimulated transcription.

Animals↗
PubMedDate not supplied

[Great potential of small RNAs: RNA interference and microRNA].

Double-stranded RNA (dsRNA) induces a sequence-specific silencing in eukaryotic cells. This silencing process beggins when long dsRNA is cleaved to 21 to 26 long small RNA by means of the RNAse III-type enzyme Dicer. These small dsRNA are included into silencing effector complexes, that are targeted to complementary sequences. Small RNA dependent gene silencing can be achieved by distinct mechanisms based depending mainly on the nature of target sequences and on the proteins present in the effector complex. The route of interference RNA (RNAi) begins when Dicer yields small interference RNA (siRNA) that bind to complementary mRNA for its degradation, forming the RISC complex. siRNA are naturally formed from transposons and dsRNA viruses during its replication, as well as from other bidirectional transcribed repetitive sequences. Some of the enzymes thar are part of the RNAi machinery, including Dicer, are encoded by multigene families in many species, that also play a role in other mechanisms of RND-dependent gene silencing. MicroRNA's (miRNA) are other small RNA's that can induce gene silencing at the mRNA level. These are formed in a general manner when Dicer process hairpin structures resulting from the transcription of non-coding sequences from plant and animal genomes. miRNA's are integrated into a RISC-like complex, after which, depending on their degree of complementarity with target mRNA, can either repress translation or induce mRNA degradation. miRNA-dependent silencing is essential for the development of multicellular organisms. Artificial RNAi induction by means of siRNA or miRNA is being used as a tool to inactivate gene expression in culture cells and in living organisms. This review focuses on the progress in the understanding of the mechanisms involved in gene regulation by RNA in animals and details some current efforts to apply theses phenomena as a tool in research and in the therapeutic of human diseases.

Humans↗
PubMed1982

Nucleotide sequences of influenza virus segments 1 and 3 reveal mosaic structure of a small viral RNA segment.

Defective interfering RNAs of influenza virus are small segments derived from viral segments 1, 2 and 3. We present here the complete nucleotide sequences of segments 1 and 3 from the human influenza strain A/PR/8/34 and deduce that the sequence of a small RNA segment from A/NT/60/68, apparently a defective interfering RNA, is derived from five separate regions in segment 3 and from one region in segment 1. These regions, which are located near the terminal of the two parental segments, are arranged in the small RNA segment in an alternating fashion: thus a region derived from near 5' terminus is adjacent to a region derived from near a 3' terminus. We propose that the small segment is generated during positive strand synthesis as a result of the viral polymerase pausing at uridine-rich sequences in the template and reinitiating synthesis at another site.

Base Sequence↗
PubMed1994

Processing of U14 small nucleolar RNA from three different introns of the mouse 70-kDa-cognate-heat-shock-protein pre-messenger RNA.

U14 is a small nucleolar RNA required for the processing of eukaryotic rRNA precursors. The U14 genes of mouse as well as rat, hamster, human, Xenopus and trout are encoded within introns of the constitutively expressed 70-kDa-cognate-heat-shock protein gene (hsc70). We demonstrate here that U14.6 and U14.8 snRNAs, in addition to the previously characterized U14.5, are processed from their respective introns when hsc70 pre-mRNA transcripts containing these intronic snRNAs are injected into Xenopus oocyte nuclei. Identical intermediates are observed in the processing of all three mouse U14 snRNAs indicating similar processing pathways. The production of U14 snRNA processing intermediates possessing either mature 5' or 3' termini demonstrated that processing can occur at either end independent of maturation at the other terminus. Processing of U14.6 from hsc70 intron 6 is not dependent upon the base pairing of intron sequences flanking the 5' and 3' termini of the encoded U14 snRNA molecule. Therefore, excision of an intronic snRNA does not require extending the 5',3' terminal helix of U14 snRNA secondary structure into flanking intron regions as originally suggested. Microinjection of the plasmid vector containing the mouse hsc70/U14.5 snRNA coding region revealed that undetermined plasmid sequences can serve as non-specific promoters to generate spurious RNA transcripts. The processing of these transcripts and examination of the plasmid-initiated transcriptional-start sites indicated that a U14-specific promoter is not present in or around the intron-encoded U14.5 gene. These results strongly suggest that biosynthesis of mouse U14 snRNA results from an intron-processing pathway.

Amino Acid Sequence↗
PubMed1994

U17XS8, a small nucleolar RNA with a 12 nt complementarity to 18S rRNA and coded by a sequence repeated in the six introns of Xenopus laevis ribosomal protein S8 gene.

U17XS8 RNA is a 220 nt small RNA coded by a sequence repeated in each of the six introns of the gene for ribosomal protein S8 of Xenopus laevis. It is mainly localized in the nucleolus, as shown by in situ hybridization, and it is assembled in a ribonucleoprotein particle (RNP) sedimenting at about 12S, slightly faster than U3 RNP, and with a density of 1.45 g/ml. DNA and RNA microinjections in Xenopus oocytes have shown that U17XS8 RNA is not the product of an independent transcription unit, but is produced by processing of intron sequences of r-protein S8 transcript, as has been recently shown for other small nucleolar RNAs encoded in the introns of other genes. Its accumulation during Xenopus development, oogenesis and embryogenesis, increases in parallel to that of r-protein S8 mRNA. Another interesting feature is the presence in the U17XS8 RNA of a 12 nt sequence complementary to 18S rRNA. The results presented suggest a possible role of this RNA in some step(s) of ribosome assembling in the nucleolus. Some relevant differences between Xenopus U17XS8 RNA and the corresponding human U17 RNA, recently described, have been observed.

Animals↗
PubMed1998

Identification and topological arrangement of Drosophila proximal sequence element (PSE)-binding protein subunits that contact the PSEs of U1 and U6 small nuclear RNA genes.

Most small nuclear RNAs (snRNAs) are synthesized by RNA polymerase II, but U6 and a few others are synthesized by RNA polymerase III. Transcription of snRNA genes by either polymerase is dependent on a proximal sequence element (PSE) located upstream of position -40 relative to the transcription start site. In contrast to findings in vertebrates, sea urchins, and plants, the RNA polymerase specificity of Drosophila snRNA genes is intrinsically encoded in the PSE sequence itself. We have investigated the differential interaction of the Drosophila melanogaster PSE-binding protein (DmPBP) with U1 and U6 gene PSEs. By using a site specific protein-DNA photo-cross-linking assay, we identified three polypeptide subunits of DmPBP with apparent molecular masses of 95, 49, and 45 kDa that are in close proximity to the DNA and two additional putative polypeptides of 230 and 52 kDa that may be integral to the complex. The 95-kDa subunit cross-linked at positions spanning the entire length of the PSE, but the 49- and 45-kDa subunits cross-linked only to the 3' half of the PSE. The same polypeptides cross-linked to both the U1 and U6 PSE sequences. However, there were significant differences in the cross-linking patterns of these subunits at a subset of the phosphate positions, depending on whether binding was to a U1 or U6 gene PSE. These data suggest that RNA polymerase specificity is associated with distinct modes of interaction of DmPBP with the DNA at U1 and U6 promoters.

Animals↗
PubMed1983

Isolation and characterization of two linked mouse U1b small nuclear RNA genes.

A 6.9 kilobase Eco R1 fragment containing genes for two U1 RNAs has been isolated from a library of mouse DNA. The two genes code for an RNA which is very similar, if not identical, to mouse U1b RNA as judged by S1 nuclease mapping. This RNA is one base longer than the mouse U1a RNA, human U1 RNA, and rat U1 RNA and differs in six nucleotide substitutions from rat U1 RNA. The two genes are five kilobases apart and the U1 RNAs are coded for on opposite strands of the DNA with the 5' ends juxtaposed. The sequences flanking the genes are identical for 700 bases 5' to the gene and at least 80 bases 3' to the gene.

Animals↗
PubMed1999

The general transcription factors IIA, IIB, IIF, and IIE are required for RNA polymerase II transcription from the human U1 small nuclear RNA promoter.

RNA polymerase II transcribes the mRNA-encoding genes and the majority of the small nuclear RNA (snRNA) genes. The formation of a minimal functional transcription initiation complex on a TATA-box-containing mRNA promoter has been well characterized and involves the ordered assembly of a number of general transcription factors (GTFs), all of which have been either cloned or purified to near homogeneity. In the human RNA polymerase II snRNA promoters, a single element, the proximal sequence element (PSE), is sufficient to direct basal levels of transcription in vitro. The PSE is recognized by the basal transcription complex SNAPc. SNAPc, which is not required for transcription from mRNA-type RNA polymerase II promoters such as the adenovirus type 2 major late (Ad2ML) promoter, is thought to recruit TATA binding protein (TBP) and nucleate the assembly of the snRNA transcription initiation complex, but little is known about which GTFs other than TBP are required. Here we show that the GTFs IIA, IIB, IIF, and IIE are required for efficient RNA polymerase II transcription from snRNA promoters. Thus, although the factors that recognize the core elements of RNA polymerase II mRNA and snRNA-type promoters differ, they mediate the recruitment of many common GTFs.

Animals↗
PubMed2004

RNA interference and the use of small interfering RNA to study gene function in mammalian systems.

In the past 2 years, extraordinary developments in RNA interference (RNAi)-based methodologies have seen small interfering RNAs (siRNA) become the method of choice for researchers wishing to target specific genes for silencing. In this review, an historic overview of the biochemistry of the RNAi pathway is described together with the latest advances in the RNAi field. Particular emphasis is given to strategies by which siRNAs are used to study mammalian gene function. In this regard, the use of plasmid-based and viral vector-based systems to mediate long-term RNAi in vitro and in vivo are described. However, recent work has shown that non-specific silencing effects and activation of the interferon response may occur following the use of some siRNA and delivery vector combinations. Future goals must therefore be to understand the mechanisms by which siRNA delivery leads to unwanted gene silencing effects in cells and, in this way, RNAi technology can reach its tremendous potential as a scientific tool and ultimately be used for therapeutic purposes.

Animals↗
PubMed1996

The sequence of 55 kb on the left arm of yeast chromosome XVI identifies a small nuclear RNA, a new putative protein kinase and two new putative regulators.

We have sequenced and analysed a 55786 bp fragment located on the left arm of chromosome XVI of Saccharomyces cerevisiae. The sequence contains 29 non-overlapping open reading frames (ORFs) longer than 300 bp, among which 12 genes have previously been sequenced: OYE3, REV3, SVS1, BEM4, CDC60, KIP2, PEP4, SPK1, PAL1, KES1, SNR17B and RPL37A. Three new ORFs, P2591, P2594 and P2597 are highly homologous to the human phosphotyrosyl phosphatase activator PTPA, to the pleiotropic regulator PRL1 of PP1 and PP2a protein phosphatases in plants and to the protein kinase PAR-1 in Caenorhabditis elegans, respectively. Three other ORFs, P2545, P2567 and P2578 have significant homology with ORFs of unknown function located on yeast chromosomes VIII, XVI and IV respectively.

Amino Acid Sequence↗