A candidate gene for human U1 RNA.

Clones containing sequences complementary to the small nuclear RNA U1 were isolated from the human DNA library of Lawn et al. (1978). Three clones were studied by hybridization and restriction enzyme cleavage. The results showed that the inserts in all three clones were different and that each clone contains one single copy of a sequence which hybridizes to U1 RNA. The results revealed moreover that only one of the three clones contains all the cleavage sites which can be predicted from the known sequence of human U1 RNA, suggesting that the three clones comprise one candidate U1 gene and two pseudogenes. A fragment from the recombinant with the candidate U1 gene was subcloned in the pPR322 plasmid and part of its sequence was determined. The results showed that the subclone contains a sequence which matches that of the human U1 RNA perfectly. The sequence "TATAT" which often is found adjacent to RNA polymerase II start sites, was identified 33-37 base pairs upstream from the beginning of the U1 sequence. Two ten base pairs long, nearly perfect, direct repeats were also identified in the vicinity of the U1 sequence and an imperfect inverted repeat follows immediately after the U1 gene.     

Mouse DNA sequences complementary to small nuclear RNA U1.

A mouse genomic library was screened for sequences complementary to U1 nuclear RNA. Out of the eight clones tested, none contained more than one copy of U1. Six of them were identical and one of those (clone 0U1-XIII) was further analyzed. This latter clone contained no other gene for discrete species of small size RNA in the 8 Kb EcoRI fragment encoding U1. A 248 bp Bg1II fragment from 0U1-XIII encompassing the full length of U1 as well as flanking regions on both sides has been subcloned and sequenced in M13 phage. Although the coding region was 96.5% homologous to rat U1a RNA, there is no direct evidence that this clone is a true gene. 3' and 5' flanking sequences of this as well as other published clones have been searched for homologies and the results of this search are discussed.     

Isolation and characterization of snRNA and scRNA gene candidates from a human genomic library

A simple procedure for the isolation and preparative gel electrophoresis of snRNA and scRNA is described. These small RNA species were used to select DNA sequences from a human genomic library which are able to protect hybridized snRNA or scRNA against T1-ribonuclease attack. The snRNA clones obtained contain only sequences for one snRNA species and only one copy of the respective gene. In contrast, more than one 7S RNA gene is present within the scRNA clones.     

A functional dominant mutation in Schizosaccharomyces pombe RNase MRP RNA affects nuclear RNA processing and requires the mitochondrial-associated nuclear mutation ptp1-1 for viability.

The essential gene for RNase MRP RNA, mrp1, was identified previously in Schizosaccharomyces pombe by homology to mammalian RNase MRP RNAs. Here we describe distinct site-specific mutations in RNase MRP RNA that support a conserved role for this ribonucleoprotein in nucleolar 5.8S rRNA processing. One characterized mutation, mrp1-ND90, displays dominance and results in accumulation of unspliced precursor RNAs of dimeric tRNA(Ser)-tRNA(Met)i, suggesting a novel nuclear role for RNase MRP in tRNA processing. Cells carrying the mrp1-ND90 mutation, in the absence of a wild-type copy of mrp1, additionally require the mitochondrially associated nuclear mutation ptp1-1 for viability. Analysis of this mrp1 mutation reinforces previous biochemical evidence suggesting a role for RNase MRP in mitochondrial DNA replication. Several mutations in mrp1 result in unusual cellular morphology, including alterated nuclear organization, and are consistent with a broader nuclear role for RNase MRP in regulating a nuclear signal for septation; these results are a further indication of the multifunctional nature of this ribonucleoprotein.     

Secondary structure of the RNA component of a nuclear/mitochondrial ribonucleoprotein.

RNase mitochondrial RNA processing (MRP) is a site-specific endoribonuclease located in both the nucleus and mitochondria of vertebrate cells. The enzyme is a ribonucleoprotein whose RNA component has been shown to be encoded by a nuclear gene. Because RNase MRP is particular in its substrate requirement, RNA-RNA interaction has been proposed as important for the cleavage reaction. A secondary structure of this RNA from mouse cells has been derived by chemical modification of in vivo MRP RNA in ribonucleoprotein form, as isolated free RNA, and as RNA synthesized in vitro. Full-length MRP RNA appears to adopt a conformation containing a significant number of single-stranded residues and may form a pseudoknot. The data are consistent with both the RNA within the ribonucleoprotein and the free RNA possessing comparable secondary structures and suggest a possible site of interaction between enzyme and substrate. The human MRP RNA can be folded into a conformation very similar to that predicted for the mouse MRP RNA. A more limited analysis of human MRP RNA is consistent with the structure proposed for the mouse species.     

Characterization of human MRP/Th RNA and its nuclear gene: full length MRP/Th RNA is an active endoribonuclease when assembled as an RNP.

Vertebrate cells contain a site-specific endoribonuclease (RNase MRP) that cleaves mitochondrial RNA transcribed from the origin of leading-strand mitochondrial DNA replication. This report presents the characterization of the human enzyme and its essential RNA component. Human RNase MRP is a ribonucleoprotein with a nucleus-encoded RNA of 265 nucleotides. As expected, the single-copy RNA coding region is homologous (84%) to the corresponding mouse gene; surprisingly, at least 700 nucleotides of the immediate 5'-flanking region are conserved. The 265-nucleotide MRP RNA and an MRP RNA cleavage product representing the 3'-terminal 108 nucleotides exist in nuclear and mitochondrial RNA isolates; the larger MRP RNA is present in greatest abundance in the nucleus. The putative processing site within the 265-nucleotide MRP RNA is offset from that of mouse MRP RNA, but in each case cleavage is precise and occurs at the sequence ANCCCGC. Oligonucleotide-mediated inhibition experiments reveal that both the 5' and 3' portions of the MRP RNA are involved in cleavage by RNase MRP; this implies that full length MRP RNA complexed with proteins is an active species in vertebrate cells.     

DNA sequences complementary to human 7 SK RNA show structural similarities to the short mobile elements of the mammalian genome

A complementary DNA clone of 7 SK RNA from HeLa cells was used to study the genomic organization of 7 SK sequences in the human genome. Genomic hybridizations and genomic clones show that 7 SK is homologous to a family of disperse repeated sequences most of which lack the 3' end of the 7 SK RNA sequence. Only few of the genomic K sequences are homologous to both 3' and 5' 7 SK probes and presumably include the gene(s) for 7 SK RNA. The sequence of four genomic 7 SK clones confirms that they are in most cases pseudogenes. Although Alu sequences are frequently found near the 3' and 5' end of K DNA, the sequences immediately flanking the pseudogenes are different in all clones studied. However, direct repeats were found flanking directly the K DNA or the K-Alu unit, suggesting that the K sequences alone or in conjunction with Alu DNA might constitute a mobile element.     

Identification of a putative RNA helicase in E.coli.

The human p68 protein, an SV40 large T related antigen, is an RNA dependent ATPase and RNA helicase. It belongs to a new large and highly conserved gene family, the DEAD box proteins, whose members are involved in a variety of processes requiring manipulation of RNA secondary structure such as translation and splicing. Multiple DEAD box genes are present in S.cerevisiae, but only one has previously been described in E.coli. Low stringency screening of an E.coli genomic library with a p68 cDNA probe led to the identification of dbpA, a new E.coli DEAD box gene located at 29.6 minutes on the W3110 chromosome. We report here the nucleotide and deduced amino acid sequences of the gene. We have overexpressed dbpA from its own promoter on a high copy number plasmid and identified the gene product as a approximately 50 kD protein by immunoblotting with an anti-DEAD antibody.     

Bovine RNase MRP cleaves the divergent bovine mitochondrial RNA sequence at the displacement-loop region

RNase MRP is a site-specific ribonucleoprotein endoribonuclease that cleaves mitochondrial RNA from the origin of leading-strand DNA synthesis contained within the displacement-loop region. Bovine mitochondrial DNA maintains the typical gene content and order of mammalian mitochondrial DNAs but differs in the nature of sequence conservation within this displacement-loop regulatory region. This markedly different sequence arrangement raises the issue of the degree to which a bovine RNase MRP would reflect the physical and functional properties ascribed to the enzymes previously characterized from mouse and human. We find that bovine RNase MRP exists as a ribonucleoprotein, with an RNA component of 279 nucleotides that is homologous to that of mouse or human RNase MRP RNA. Characterization of the nuclear gene for bovine RNase MRP RNA showed conservation of sequence extending 5 of the RNase MRP RNA coding sequence, including the presence of a cis-acting element known to be important for the expression of some mitochondrial protein-coding nuclear genes. Bovine or mouse RNase MRP cleaves a standard mouse mitochondrial RNA substrate in the same manner; each also cleaves a bovine mitochondrial RNA substrate identically. Since bovine and mouse RNase MRPs process both bovine and mouse substrates, we conclude that the structural features of the mitochondrial RNA substrate required for enzymatic cleavage have been well conserved despite significant overall primary sequence divergence. Inspection of the bovine RNA substrate reveals conservation of only the most critical portion of the primary sequence as indicated by earlier studies with mouse and human RNase MRPs. Interestingly, a principal cleavage site in the bovine mitochondrial RNA substrate is downstream of the promoter located at the leading-strand mitochondrial DNA replication origin. Correspondence to: D.J. Dairaghi     

The 40-kilodalton to autoantigen associates with nucleotides 21 to 64 of human mitochondrial RNA processing/7-2 RNA in vitro.

A 40-kDa To antigen recognized by sera from some patients with autoimmune diseases is an integral component of both human RNase P and mitochondrial RNA processing (MRP) RNase. Human MRP and RNase P RNAs, synthesized in vitro, readily associate with the To antigen present in the HeLa cell extract. Using this in vitro reconstitution system, the binding site of the To antigen is localized to a 44-nucleotide-long sequence corresponding to nucleotides 21 to 64 of the human MRP RNA. UV cross-linking experiments showed that the To antigen binds directly to MRP RNA and to RNase P (H1) RNA through RNA-protein interactions. Although the MRP RNA and RNAse P (H1) RNA show sequence homology in four conserved blocks (H. A. Gold, J. N. Topper, D. A. Clayton, and J. Craft, Science 245:1377-1380, 1989), the To antigen-binding site in MRP RNA does not show any obvious primary sequence homology with H1 RNA. These data suggest that the To antigen binds to a conserved and presumably a common secondary or tertiary structure in human MRP and RNase P RNAs.