R Reddy

Pseudouridine residues in the 5′-terminus of uridine-rich nuclear RNA I (U1 RNA)

Abstract The primary nucleotide sequence was reported earlier for U1 RNA (Reddy et al, (1974) J. Biol. Chem. 249 , 6486–6494), an snRNA implicated in splicing of HnRNAs. In view of the presence of homologous pseudouridine (ψ) residues in 5′-ends of several highly conserved U-snRNAs and the recent report of modified bases in the U1 RNA structure (Branlant et al, (1980) Nucleic Acids Res. 8 , 4143–4154) a study was made for the presence of ψ and other modified nucleotides in the 5′-end of the U1 RNA. Identification of ψ residues at positions 6 and 7, shows the 5′-sequence of U1 RNA is: m 3 2 , 2,7 GpppAm-Um-A-C-ψ-ψ-A-C-C-U-G-G-C-A-G-G-G-G-A-G-A-U-A-C. The ψ residues in place of U at positions 6 and 7 may affect the binding of U1 RNA at intron-exon splice junctions.

5S RNAIII, a new nucleus-specific 5S RNA

A new species of 5S RNA, referred to as 5S RNAIII, has been found in the Novikoff hepatoma ascites cell nuclei. This RNA, which composes 28% of total nuclear 5S RNA and co-migrates with ribosomal 5S RNAI and 5S RNAII on 8% polyacrylamide gel electrophoresis, has been isolated by chromatography on DEAE Sephadex columns. The nucleotide composition is A 24, U 31, G 24 and C 20; its A+UG+C ratio is 1.24. T1 RNase digestion produced the following unique oligonucleotides from 5S RNAIII: (U2C2)Gp, (CUA)Gp, (C4A3U8)Gp, [(UmU) (C2A3U5) (AGmC)], (CAU2)Gp, (C2AU2)Gp and (C3A3U2)Gp. Similarly, unique oligonucleotides were produced by complete pancreatic RNase digestion of 5S RNAIII, including UmUp, A-A-Up, A-A-Cp, (AG)Cp, A-Gm-Cp, (A2G)Up, A-A-A-Up and (A3G)-Up.

Identification of three different anti-4S RNA sera associated with autoimmune disease

Abstract Analysis of antibodies obtained from 10 patients with myositis, 20 with systemic lupus erythematosus and 24 with scleroderma revealed that 2 patients with myositis and one with systemic lupus erythematosus produced antibodies precipitating 4S RNAs. Each of the 3 anti-4S RNA sera precipitated a different subset of the total 4S RNA population. Two sera precipitated deproteinized or gel extracted 4S RNA from Novikoff hepatoma or Dinoflagellate cells. The other serum did not precipitate deproteinized RNA and may precipitate a 4S RNA in a small RNP particle. The reactive 4S RNAs are extensively modified, which suggests that they may be tRNAs, tRNA precursors or previosly unknown RNA species.

Two-dimensional polyacrylamide gel electrophoresis separation of low molecular weight nuclear RNA.

Summary Two-dimensional electrophoresis successively on 10%, pH 7.2, and 12%, pH 3.3, polyacrylamide gels was used to separate nuclear and whole cell 4–8S RNA fractions from Novikoff hepatoma ascites cells into individual RNA species. With this method, analytical studies were possible on two new species of RNA referred to as 4.2S RNA and 4.5S RNAII, respectively. The 4.2S RNA has the nucleotide composition AMP, 18.0; UMP, 23.9; GMP, 27.9; and CMP 26.7. This RNA contains 2 ψMP residues and an alkali stable dinucleotide. The 4.5S RNAII was distinctly separated from 4.5S RNAI and 4.5S RNAIII and has the nucleotide composition AMP, 20.6%; UMP, 23.7%; GMP, 30.0%; and CMP, 25.7%. It has no ψMP or 2′-0-methylated nucleotides. In addition, several other spots were separated from the major RNA species.

Low molecular weight nuclear RNA. The 3′-terminal sequence of the U2 RNA

SummaryThe U2 RNA is one of the low molecular weight nuclear RNAs of Novikoff hepatoma ascites cells that is specifically localized in the extranucleolar portion of the nucleus. After the RNA was purified by polyacrylamide gel electrophoresis, it was subjected to various enzymatic digestion procedures for determination of its primary sequence of nucleotides. The U2 RNA contains 197 nucleotides. Its 5′ end, which is highly modified, contains N2, 2, 7-trimethyl guanylic acid and 11 pseudouridylic acid residues (Reddy et al, J. Biol. Chem. 247, 7245–7250, 1972) in addition to 10 2′-O-methyl ribose residues.Its 3′-terminal portion (128 nucleotides) contains no methylated nucleotides and its sequence is:-A-U-A-C-G-U-C-C-U-C-U-A-U-C-C-G-A-G-G-A-C-A-A-U-A-ψ-U-A-ψ-U-A-A-A-U-G-G-A-U-U-U-U-U-G-G-A-A-C-U-A-G-G-A-G-U-U-G-G-A-A-U-A-G-G-A-G-C-U-U-G-C-U-C-C-G-U-C-C-A-C-C-U-C-A-C-G-C-A-U-C-G-A-C-C-U-G-G-U-A-U-U-G-C-G-C-A-G-U-A-C-C-C-U-C-A-G-G-A-A-C-G-G-U-G-C-A-C-C-AO H.

Small nuclear RNPs and RNA processing

ABSTRACT Small RNAs discovered about 16 years ago are metabolically stable, conserved through evolution, and localized to specific subcellular compartments. The sequencing of small RNAs has resulted in the discovery of cap structures, and the nucleotide sequences of all the six capped snRNAs are defined, in some cases for several species. The discovery that patients with autoimmune diseases produce antibodies directed against small RNPs, and the hypothesis that U1 RNA may be involved in splicing hnRNAs, has brought small RNAs to the attention of many investigators. All the capped snRNAs appear to be synthesized by polymerase II, and other small RNAs by polymerase III. Genes having identical sequences to U1, U2, and U3 RNA have been isolated and, interestingly, the human genome appears to contain more pseudogenes for small RNAs than real genes.

Methylation of snRNAs and 18S rRNA of Novikoff heptoma

In the biosynthesis of mature snRNAs and rRNAs, RNA methylation is one of the important mechanisms involved in their posttranscriptional modifications. The processes of RNA methylation are catalyzed by RNA methylases which specifically recognize methylation sites. This enzymatic recognition of methylating sites is specific for particular species of RNA and dictates the types, numbers of methylated nucleotide sequences and their order in maturation stages (Perry, 1976).

Structures of the U-snRNAs

The work of our laboratory in 1962 led to the evolution of methods for isolation of nucleoli (Busch and Smetana 1970). In the course of these studies it became apparent that the nucleoli were very complex structures that contained a variety of enzymes, nonhistone proteins, the rDNA and precursors of rRNA. It was not known at that time that in addition to many species of macromolecules involved in ribosome synthesis, nucleoli also contained slowly turning over elements.