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.     

Cloning and characterization of cDNA copies of the 7S RNAs of HeLa cells

We have cloned cDNA copies of in vitro adenylated 7S RNA of HeLa cells. The most representative clones in the library contain DNA fragments copied from the 7SL and 7SK small RNAs. The two classes of recombinants share no homology. The 7SL RNA contains at the 5' end of the molecule sequences homologous to the Alu sequence family. Hybridization to human genomic DNA shows that the 7SL and 7SK clones are homologous to two different families of repetitive sequences.     

Methylphosphate cap structure increases the stability of 7SK, B2 and U6 small RNAs in Xenopus oocytes.

We studied the role of the methylphosphate cap structure in the stability and nucleocytoplasmic transport by microinjecting U6, 7SK and B2 RNAs into the Xenopus oocytes. In every case, the methylphosphate capped RNAs were 3 to 9 times more stable than the uncapped RNAs. When a methylphosphate cap structure was placed on human H1 RNA which is normally not capped, its stability was improved 2-7 fold. These data show that the methylphosphate cap enhances the stability of 7SK, B2, H1 and U6 RNAs. The methylphosphate-capped 7SK RNA was transported into the nucleus from cytoplasm, but remained in the nucleus when injected into the nucleus; in this respect, 7SK RNA exhibited properties previously shown for U6 RNA. Both U6 and 7SK RNAs with ppp on their 5' ends were transported from cytoplasm to the nucleus suggesting that the methylphosphate cap structure is not required for transport of these RNAs across the nuclear membrane.     

The conserved 7SK snRNA gene localizes to human chromosome 6 by homolog exclusion probing of somatic cell hybrid RNA.

Many small RNAs contribute essential activities to eukaryotic cells. In mammalian genomes dispersed repetitive sequences which exhibit homology to small RNAs often exist as pseudogenes which can complicate identification, localization, and analysis of the authentic gene. We mapped a productive human 7SK small nuclear RNA gene to human chromosome 6 by analyzing Northern blots derived from a panel of somatic cell hybrids that contain single human chromosomes. In order to avoid crossreactivity of the probe with rodent 7SK RNA, which is 98% identical to human 7SK, a method termed homolog exclusion probing was developed. This method uses an excess of non-labelled rodent-specific oligodeoxynucleotide to block the rodent 7SK RNA from hybridizing with the human-specific oligodeoxynucleotide probe. The effectiveness of this method to enhance the human 7SK RNA signal is demonstrated. The potential to map and subsequently isolate other small RNA genes by this approach and the use of homolog exclusion probing to discriminate among family members of highly related RNAs and DNAs in a single species is discussed.     

U4 small nuclear RNA pseudogenes from rat genome have common truncated 3'-ends

Four U4 RNA pseudogenes were isolated and characterized from a rat genomic bank. The four pseudogenes contained sequences completely homologous to U4 RNA from nucleotides 1 to 67 and had common truncated 3'-ends. Three of the four pseudogenes were flanked by 14 to 18 nucleotide-long direct repeats. The structural features of these four U4 RNA pseudogenes are consistent with the hypothesis that these pseudogenes arose by RNA self-primed complementary DNA synthesis and integration into the genome (Van Arsdell et al., Cell 26:11-17, 1981).     

Structural analyses of the 7SK ribonucleoprotein (RNP), the most abundant human small RNP of unknown function.

The human 7SK ribonucleoprotein (RNP) has been analyzed to determine its RNA secondary structure and protein constituents. HeLa cell 7SK RNA alone and within its RNP have been probed by chemical modification and enzymatic cleavage, and sites of modification or cleavage have been mapped by primer extension. The resulting secondary structure suggests that structural determinants necessary for capping (a 5' stem followed by the sequence AUPuUPuC) and nuclear migration (the sequence AUPuUPuC) of 7SK RNA may be similar to those for U6 small nuclear RNA (snRNA). It also supports existence of a 3' stem structure which could serve to self-prime cDNA synthesis during pseudogene formation. Oligonucleotide-directed RNase H digestion indicated regions of 7SK RNA capable of base pairing with other nucleic acids. Antisense 2'-O-methyl RNA oligonucleotides were used to affinity select the 7SK RNP from an in vivo 35S-labeled cell sonic extract and identify eight associated proteins of 83, 48, 45, 43, 42, 21, 18, and 13 kDa. 7SK RNA has extensive sequence complementarity to U4 snRNA, within the U4/U6 base pairing domain, and also to U11 snRNA. The possibility that the 7SK RNP is an unrecognized component of the pre-mRNA processing machinery is discussed.     

Cloning and characterisation of the abundant cytoplasmic 7S RNA from mouse cells.

A cDNA library has been prepared from mouse embryo small RNAs and screened for the presence of clones complementary to the highly abundant cytoplasmic 7S RNA. One clone (pA6) was selected which hybridized exclusively with 7S RNA on a Northern blot prepared from cytoplasmic RNA run on high resolution polyacrylamide/urea gels. Sequence analysis of this clone has shown that at least 65 nucleotides at the 5' end of 7S RNA are extensively homologous with the highly repeated mouse B1 family. Heterologous hybridisations between the cloned mouse 7S sequence and RNAs prepared from rat, human and chick cells have shown that the non-B1 part of the 7S RNA molecule has been highly conserved during recent eucaryotic evolution. There are multiple copies of 7S RNA genes in the genomes of mouse, human, rat and chick cells, but substantial differences exist in copy number and genomic organisation in these organisms.     

Characterization of U6 small nuclear RNA cap-specific antibodies. Identification of gamma-monomethyl-GTP cap structure in 7SK and several other human small RNAs

The cap structure in human U6 small nuclear (sn)RNA, gamma-monomethylguanosine triphosphate (meGTP), was conjugated to human serum albumin and used as antigen to raise polyclonal antibodies in rabbits. The resulting antibodies reacted specifically with meGTP but not with GTP, GDP, GMP, meGMP, meATP, meCTP, meUTP, or with methyl phosphate in enzyme-linked immunosorbent assay and/or in radioimmunoassays. Although less efficiently, meGDP was also recognized by these antibodies. Indirect immunofluorescence studies with anti-meGTP antibodies showed predominantly nuclear immunofluorescence. Anti-meGTP antibodies immunoprecipitated intact U6 snRNA from a mixture of HeLa cell RNAs. In addition to the U6 snRNA, anti-meGTP antibodies immunoprecipitated several additional small RNAs that varied in length from approximately 50 to 330 nucleotides. These RNAs contained the meGTP cap structure and are structurally distinct from U6 snRNA. One of these meGTP-containing RNAs was found to be previously characterized 7SK RNA; human 7SK RNA synthesized in vitro also contained the same cap structure. Results obtained in this study provide evidence for the presence of gamma-monomethyl-GTP cap structure in a wide spectrum of human cellular RNAs. These antibodies will be useful in studying the structure and function of this new family of small RNAs.     

cDNA genes formed after infection with retroviral vector particles lack the hallmarks of natural processed pseudogenes.

Retroviral proteins can encapsidate RNAs without retroviral cis-acting sequences. Such RNAs are reverse transcribed and inserted into the genomes of infected target cells to form cDNA genes. Previous investigations by Southern blot analysis of such cDNA genes suggested that they were truncated at the 3' and the 5' ends (R. Dornburg and H. M. Temin, Mol. Cell. Biol. 8:2328-2334, 1988). To analyze such cDNA genes further, we cloned three cDNA genes (derived from a hygromycin B phosphotransferase gene) in lambda vectors and analyzed them by DNA sequencing. We found that they did not correspond to the full-length mRNA: they were truncated at both the 3' and the 5' ends, did not contain a poly(A) tract, and were not flanked by direct repeats. The 3'-end junctions to chromosomal DNA of five more cDNA genes were amplified by polymerase chain reaction, cloned in pUC vectors, and sequenced. All of these cDNA genes had 3'-end truncations, and no poly(A) tracts were found. Further polymerase chain reaction experiments were performed to detect hygromycin B phosphotransferase cDNA genes with a poly(A) tract in DNA extracted from a pool of about 500 colonies of cells containing cDNA genes. No hygromycin B phosphotransferase cDNA gene with a poly(A) tract was found. Investigation of two preintegration sites by Southern analysis revealed that deletions were present in chromosomal DNA at the site of the integration of the cDNA genes. Naturally occurring processed pseudogenes correspond to the full-length mRNA, contain a poly(A) sequence, and are flanked by direct repeats. Our data indicate that cDNA genes formed by infection with retrovirus particles lack the hallmarks or natural processed pseudogenes. Thus, it appears that natural processed pseudogenes were not generated by retrovirus proteins.