Position of the rearranged V kappa and its 5' flanking sequences determines the location of somatic mutations in the J kappa locus

Somatic hypermutation is known to occur in the VJ kappa exon and its flanking sequences, yet little is known about the hypermutation mechanism or its exact target within the rearranged locus. Mutations may occur at the same frequency, spanning a region from the leader intron to 3' of J kappa 5, regardless of which J is chosen for VJ rearrangement. Another possibility is that mutations may be limited to the rearranged VJ kappa and its immediate flanking sequences. To distinguish between these possibilities, the JC introns of 21 alleles with V kappa rearranged to J kappa 1 were sequenced, and mutations were located. The frequency of mutations was determined for different sections of the intron and compared with the frequencies of mutations found in the JC intron of a set of VJ kappa 5 alleles. The results showed that mutations were concentrated in and around the rearranged VJ, regardless of whether J kappa 1 or J kappa 5 was used. These data imply that the hypermutational mechanism focuses on rearranged V genes.     

p55 IL-2 receptor mRNA precursors in murine T lymphocyte nuclei

An unusual family of cDNA clones homologous to human p55 IL-2R sequences was isolated from the murine HT-2 Th cell line. These clones were mapped, partially sequenced, and compared with previously published human and mouse IL-2R sequences. They appear to consist of various combinations of exons and introns, suggesting that they are derived from p55 IL-2R mRNA precursors. The configuration of exons in the splicing intermediates indicates that the murine and human gene organizations are similar and that the 3' end of intron 3 is well conserved between the two species. RNA mapping experiments using nuclear, cytoplasmic, and total RNA and probes derived from various parts of the p55 IL-2R gene support and extend the sequence data. They indicate that detectable amounts of immature p55 IL-2R mRNA are found specifically in the cell nucleus of the HT-2 cell line. Similar data were obtained for the Th cell clone 52.3 and the cytotoxic T cell line CTLL. All these results indicate that the T cell nucleus contains significant amounts of immature p55 IL-2R mRNA.     

Nuclei of adenovirus 2-infected cells contain an RNA species that corresponds to an intron excised intact from mRNA precursors.

We used Northern and S1 nuclease analyses to identify a nuclear RNA species of the structure predicted for an intron excised from the precursor of adenovirus 2 E2A early mRNA. The structure of this excised intron demonstrated that the splicing of E2A mRNA can involve the removal of the intron sequences as single intact molecules. The concentration of the excised intron is 30 copies per nuclei, comparable to the levels of E2A polyadenylated splicing precursors, but 30-fold less than nuclear E2A mRNA. Late in infection, when the production of E2A early mRNA is dramatically elevated (Goldenberg et al., J. Virol. 38:932-939, 1981), the excess intron was not detected, indicating that either its stability or the mechanism of splicing is altered. We also identified a spliced nonpolyadenylated E2A nuclear RNA species with a structure similar to the mRNA, indicating that splicing may normally occur in the absence of polyadenylation.     

RNA metabolism in nuclei: selective transport of kappa exons from myeloma nuclei and adenoviral transcripts from infected HeLa nuclei.

Two independent systems and several analytical procedures have been used to establish that isolated mammalian nuclei selectively transport mature RNA polymerase I and II products. Murine myeloma nuclei retain physiologic restriction in our transport assay as assessed by the transport of the immunoglobulin kappa light chain mRNA and 18S and 28S rRNAs. Nearly 50% of the total kappa exons are transported as structurally intact mature mRNA molecules while less than 8% of either pulse-labeled or steady state kappa intron sequences are detected in the transported fraction. Ribosomal external transcribed spacer sequences also are absent in transported RNA. Release of cytoplasmic RNA from the outer nuclear membrane during the transport assay accounts for less than 10% of transported RNA. Nuclei isolated from adenovirus-infected HeLa cells at 20 hours post infection retain cellular actin mRNA and transport viral poly A+RNA. Ribosomal RNA is transported from infected nuclei although at a reduced rate compared to transport from mock-infected nuclei. Inhibition of transport of host mRNA is paralleled by the absence of pulse-labeled actin mRNA in the cytoplasm of infected cells. The implications of our transport data in relationship to intranuclear RNA trafficking are discussed.     

Sequences within the last intron function in RNA 3''-end formation in cultured cells.

In cultured cells, little if any mRNA accumulates from an intronless version of the human gene for triosephosphate isomerase (TPI), a gene that normally contains six introns. By deleting introns either individually or in combinations, it was demonstrated by Northern (RNA) blot hybridization that while the deletion of a greater number of introns generally results in a lower level of product mRNA, not all introns contribute equally to mRNA formation. For example, intron 1 appeared to be dispensable, at least when the remaining introns are present, but deletion of the last intron, intron 6, reduced the level of product mRNA to 51% of normal. To determine how intron 6 contributes to mRNA formation, partial deletions of intron 6 were constructed and analyzed. Deletion of the lariat and acceptor splice sites or the donor, lariat, and acceptor splice sites, each of which precluded removal of the intron 6 sequences that remained, reduced the level of product mRNA to < 1 or 27% of normal, respectively. As measured by RNase mapping and cDNA sequencing, the decrease in mRNA abundance that was attributable to the complete and partial intron 6 deletions was accompanied by an increase in the abundance of pre-mRNA that lacked a mature 3' end, i.e., that was neither cleaved nor polyadenylated. We infer from these and other data that sequences within the final intron facilitate proper 3'-end formation, possibly through an association with the components of a productive spliceosome.     

Correlation between splicing sites within an intron and their sequence complementarity with U1 RNA

We have determined the nucleotide sequence of two short introns (respectively 215 and 90 nucleotides) in the chick alpha 2-collagen (type I) gene as well as parts of the adjacent exons. For one of these introns we find that the 5' end of U1 RNA is complementary not only to the two ends of the intron but also to one end of the intron and sequences inside this intron. These complementarities predict three potential internal splicing sites. By S1 mapping experiments we find three discrete RNA precursors in which different portions of this intron have been deleted. The sizes of the deleted segments are in good agreement with the location of the predicted splicing points inside the intron. The DNA sequence indicates that removal of one portion of the intron should still allow the subsequent elimination of the rest of the intron and the correct splicing of the coding segments located at each end of the intron. The new introns created by the first splicing events contain sequences at each end which are also complementary to U1 RNA. Our data indicate that in the intron which we have examined the sequences at the 3' end of the intron are removed before those at the 5' end.