Abundant Small RNAs Research Articles

Primer design for a prokaryotic differential display RT-PCR.

We have developed a primer set for a prokaryotic differential display of mRNA in the Enterobacteriaceae group. Each combination of ten 10mer and ten 11mer primers generates up to 85 bands from total Escherichia coli RNA, thus covering expressed sequences of a complete bacterial genome. Due to the lack of polyadenylation in prokaryotic RNA the type T11VN anchored oligonucleotides for the reverse transcriptase reaction had to be replaced with respect to the original method described by Liang and Pardee [ Science , 257, 967-971 (1992)]. Therefore, the sequences of both the 10mer and the new 11mer oligonucleotides were determined by a statistical evaluation of species-specific coding regions extracted from the EMBL database. The 11mer primers used for reverse transcription were selected for localization in the 3'-region of the bacterial RNA. The 10mer primers preferentially bind to the 5'-end of the RNA. None of the primers show homology to rRNA or other abundant small RNA species. Randomly sampled cDNA bands were checked for their bacterial origin either by re-amplification, cloning and sequencing or by re-amplification and direct sequencing with 10mer and 11mer primers after asymmetric PCR.

A Novel Protein Encoded by Small RNAs of Parvovirus B19

A second small ORF contained within the parvovirus B19 genome has been shown to direct synthesis of a small 7.5-kDa protein. These results extend our previous studies in which we identified a family of 11-kDa proteins (J. St. Amand, C. Beard, K. Humphries, and C. R. Astell, Virology 183, 133-142,1991; J. St. Amand and C. R. Astell, Virology, 192, 121-131, 1993). The presumed ORF encoding the 7.5-kDa protein was discovered on the left side of the viral genome and was identified by expression of the protein and in vitro mutagenesis. Two small RNA transcripts of B19 virus were demonstrated to be able to direct the synthesis of this 7.5-kDa protein by in vitro translation using rabbit reticulocyte lysates. Expression of 7.5-kDa protein was also observed in COS-7 cells transfected with a plasmid containing the B19 genome as well as in human peripheral mononucleocytes infected with B19 virus. In both cases the 7.5-kDa protein was detected by immunoprecipitation of cell lysates with polyclonal antibodies raised against a thetic peptide corresponding to a portion of the predicted protein sequence. These data partially reveal the function the abundant small RNA transcripts produced by parvovirus B19.

Cap structure of U3 small nucleolar RNA in animal and plant cells is different. gamma-Monomethyl phosphate cap structure in plant RNA.

U3 small nucleolar RNA (snoRNA) is an abundant small RNA involved in the processing of pre-ribosomal RNA of eukaryotic cells. U3 snoRNA has been previously characterized from several sources, including human, rat, mouse, frog, fruit fly, dinoflagellates, slime mold, and yeast; in all these organisms, U3 snoRNA contains trimethylguanosine cap structure. In all instances where investigated, the trimethylguanosine-capped snRNAs including U3 snoRNA, are synthesized by RNA polymerase II. However, in higher plants, the U3 snoRNA is synthesized by RNA polymerase III and contains a cap structure different from trimethylguanosine (Kiss, T., and Solymosy, F. (1990) Nucleic Acids Res. 18, 1941-1949; Marshallsay, C., Kiss, T., and Filipowicz, W. (1990) Nucleic Acids Res. 18, 3451-3458; Kiss, T., Marshallsay, C., and Filipowicz, W. (1991) Cell 65, 517-526). In this study, we present evidence that cowpea and, most likely, tomato plant U3 snoRNA contains a methyl-pppA cap structure. These data show that the same U3 snoRNA contains different cap structures in different species and suggest that the kind of cap structure that an uridylic acid-rich small nuclear RNA contains is dependent on the RNA polymerase responsible for its synthesis. In vitro synthesized plant U3 snoRNA, with pppA or pppG as its 5' end, was converted to methyl-pppA/G cap structure in vitro when incubated with extracts prepared from wheat germ or HeLa cells. These data show that the capping machinery is conserved in organisms as evolutionarily distant as plants and mammals. Nucleotides 1-45 of tomato U3 snoRNA, which are capable of forming a stem-loop structure, are sufficient to direct the methyl cap formation in vitro.

Fractionation of HeLa cell nuclear extracts reveals minor small nuclear ribonucleoprotein particles.

Upon chromatographic fractionation of HeLa cell nuclear extracts, small RNAs of 145 and 66/65 nucleotides, respectively, were detected that are distinct from the abundant small RNAs present in the extract. These RNAs are precipitated by antibodies directed against the trimethylguanosine cap structure, characteristic for small nuclear RNAs (snRNAs) of the U type. The RNAs of 145 and 66/65 nucleotides appear to be associated with at least one of the proteins common to the major small nuclear ribonucleoprotein particles U1 to U6, since they are specifically bound by anti-Sm antibodies. These criteria characterize the RNAs that are 145 and 66/65 nucleotides in length as U-type snRNAs. Upon gel filtration, the RNAs are found within particles of molecular weights approximately equal to 150,000 and 115,000, respectively. The RNA of 145 nucleotides represents a different minor snRNA, designated U11, whereas the RNA of 66/65 nucleotides may correspond to either mammalian U7 or U10 RNA.