Identification of the major spliceosomal RNAs in Dictyostelium discoideum reveals developmentally regulated U2 variants and polyadenylated snRNAs

Most eukaryotic mRNAs depend upon precise removal of introns by the spliceosome, a complex of RNAs and proteins. Splicing of pre-mRNA is known to take place in Dictyostelium discoideum, and we previously isolated the U2 spliceosomal RNA experimentally. In this study, we identified the remaining major spliceosomal RNAs in Dictyostelium by a bioinformatical approach. Expression was verified from 17 small nuclear RNA (snRNA) genes. All these genes are preceded by a putative noncoding RNA gene promoter. Immunoprecipitation showed that snRNAs U1, U2, U4, and U5, but not U6, carry the conserved trimethylated 5' cap structure. A number of divergent U2 species are expressed in Dictyostelium. These RNAs carry the U2 RNA hallmark sequence and structure motifs but have an additional predicted stem-loop structure at the 5' end. Surprisingly, and in contrast to the other spliceosomal RNAs in this study, the new U2 variants were enriched in the cytoplasm and were developmentally regulated. Furthermore, all of the snRNAs could also be detected as polyadenylated species, and polyadenylated U1 RNA was demonstrated to be located in the cytoplasm.     

The structure of the human apolipoprotein C-II gene. Electron microscopic analysis of RNA:DNA hybrids, complete nucleotide sequence, and identification of 5' homologous sequences among apolipoprotein genes

Cloned human apo-C-II cDNA was used as a hybridization probe to identify the human apo-C-II gene in a genomic library constructed in our laboratory. The isolated apo-C-II DNA was studied both by electron microscopy and by direct sequence analysis. Ultrastructural morphological analysis of RNA-DNA hybrids revealed that the apo-C-II gene had complex structures because of regions of inverted complementary sequences in and around the gene forming stem-and-loop structures which interfere with the formation of stable RNA:DNA hybrids. Extensive morphological analysis revealed a minimum of 3 intervening sequences (IVS), and their lengths were measured. Direct sequence analysis of the cloned gene confirmed the presence of 3 IVS. There are 4 Alu type sequences in IVS-I. We sequenced 4340 nucleotides which include 545 nucleotides in the 5' flanking region, the entire gene which spans 3320 nucleotides, and 475 nucleotides in the 3' flanking region which also encompasses an additional Alu sequence. The 5' end of the gene was identified by primer extension and sequencing of the primer extended cDNA. Apo-C-II mRNA structure was deduced from the cDNA sequence, the primer extension experiments, and the genomic sequence. It is 494 nucleotides in length. Its sequence differs from previously published sequences in that there are 7 additional nucleotides before the polyadenylate tail. In the 5' flanking region, nucleotides -234 to -213 encompass a GC-rich region which exhibits high homology (greater than 70%) to the 5' flanking regions of the genes of all the apolipoproteins published to date, namely, apo-A-II (-497 to -471), apo-A-I (approximately -196 to -179), apo-E (-409 to -391), and apo-C-III (approximately -116 to -103). This highly conserved region might represent some evolutionarily conserved sequences from these related genes and/or might represent a region with regulatory function.     

Three pseudogenes for human U13 snRNA belong to class III.

The nucleotide sequences of three pseudogenes for the small nucleolar RNA, U13, were determined from three human DNA clones. The sequences are reported 50 bp 5' and 3' to each gene. These pseudogenes belong to class III because they contain dispersed mismatches when compared to the previously determined U13 RNA sequence, an adenine-rich region at the 3' end, and short imperfect repeats flanking the 5' end of the coding sequence and the 3' end of the adenine-rich region.     

Structural and functional analysis of a growth-regulated gene, the human calcyclin

Calcyclin was originally defined as a cDNA clone (2A9) whose cognate RNA is growth-regulated and whose sequence shows strong similarities to the sequences of the S-100 protein, a calcium-binding protein, as well as to a subunit of the major cellular substrate for tyrosine kinase. Using the full-length cDNA, we have now isolated from a human genomic library several phages containing calcyclin sequences. One of the phages, ch. 28-10, contains the entire calcyclin gene, plus extensive flanking sequences. The calcyclin gene is a unique copy gene and has 3 exons. The 5' flanking sequence has been characterized, both structurally and functionally. Besides a TATA box, it contains, in the region proximate to the cap site, GC boxes and a sequence with a strong homology to the enhancer core of the SV40 promoter. Other enhancer-like elements are found scattered in both the 5' and 3' flanking regions. The proximate 5' flanking region is very active in driving the transient expression of linked reporters in transfection experiments. Finally, the calcyclin gene has been localized to the long arm of human chromosome 1, near the ski oncogene.     

Spliceosomal snRNAs in the unicellular eukaryote Trichomonas vaginalis are structurally conserved but lack a 5'-cap structure

Few genes in the divergent eukaryote Trichomonas vaginalis have introns, despite the unusually large gene repertoire of this human-infective parasite. These introns are characterized by extended conserved regulatory motifs at the 5' and 3' boundaries, a feature shared with another divergent eukaryote, Giardia lamblia, but not with metazoan introns. This unusual characteristic of T. vaginalis introns led us to examine spliceosomal small nuclear RNAs (snRNAs) predicted to mediate splicing reactions via interaction with intron motifs. Here we identify T. vaginalis U1, U2, U4, U5, and U6 snRNAs, present predictions of their secondary structures, and provide evidence for interaction between the U2/U6 snRNA complex and a T. vaginalis intron. Structural models predict that T. vaginalis snRNAs contain conserved sequences and motifs similar to those found in other examined eukaryotes. These data indicate that mechanisms of intron recognition as well as coordination of the two catalytic steps of splicing have been conserved throughout eukaryotic evolution. Unexpectedly, we found that T. vaginalis spliceosomal snRNAs lack the 5' trimethylguanosine cap typical of snRNAs and appear to possess unmodified 5' ends. Despite the lack of a cap structure, U1, U2, U4, and U5 genes are transcribed by RNA polymerase II, whereas the U6 gene is transcribed by RNA polymerase III.     

trans-splicing to spliceosomal U2 snRNA suggests disruption of branch site-U2 pairing during pre-mRNA splicing

Pairing between U2 snRNA and the branch site of spliceosomal introns is essential for spliceosome assembly and is thought to be required for the first catalytic step of splicing. We have identified an RNA comprising the 5' end of U2 snRNA and the 3' exon of the ACT1-CUP1 reporter gene, resulting from a trans-splicing reaction in which a 5' splice site-like sequence in the universally conserved branch site-binding region of U2 is used in trans as a 5' splice site for both steps of splicing in vivo. Formation of this product occurs in functional spliceosomes assembled on reporter genes whose 5' splice sites are predicted to bind poorly at the spliceosome catalytic center. Multiple spatially disparate splice sites in U2 can be used, calling into question both the fate of its pairing to the branch site and the details of its role in splicing catalysis.