Loci for human U1 RNA: structural and evolutionary implications

Three clones U1-1, U1-6, and U1-8 containing sequences related to human U1 RNA have been studied by sequence analysis. The results show that each of the three clones represents a distinct locus. The U1-6 locus is closely related to the HU1-1 locus, which is believed to represent a functional U1 gene. The U1-1 and U1-8 loci are pseudogenes by definition, since they contain sequences that are closely related to but not identical with the human U1 RNA sequence. The U1-6 locus contains the sequence T-A-T-A-T close to the 5'-end of the U1 sequence but it is unclear if this represents the promoter. When the U1-8 locus was compared to the U1-6 locus, it was observed that the 5'-flanking sequences, except in the immediate vicinity of the pseudogene, are as well-conserved as the U1-related sequence itself, at least up to position -220. The high degree of homology in the 5'-flanking region suggests that U1 genes have a much more strict sequence requirement with regard to 5'-flanking sequences than most other eukaryotic genes. The U1-6 and U1-8 loci contain the sequence T-A-T-G-T-A-G-A-T-G-A between positions -211 and -221. An identical sequence is present in the equivalent position in the HU1-1 locus, and may represent the promoter. The high degree of conservation in the postulated promoter region indicates that pseudogenes like U1-8 possibly could be expressed. A truncated U1-related sequence is present between 106 to 150 nucleotides upstream from the U1 gene/pseudogene in the U1-6, the U1-8 and the HU1-1 loci, suggesting that the U1 genes may have been clustered early in evolution. The U1-1 locus has a strikingly different structure from the U1-8 locus; the pseudogene itself is as closely related to the U1 RNA sequence as is the U1-8 pseudogene but the flanking sequences, both on the 5' and the 3' side, share no detectable homology with the corresponding regions in the U1-6 or U1-8 loci. It may therefore be postulated that small nuclear RNA pseudogenes are created by several different mechanisms.     

Structural analysis of gene loci for rat U1 small nuclear RNA.

Four phage clones which hybridize with U1 small nuclear RNA were obtained from a rat gene library. Two clones contain a presumed pseudogene. A third clone includes two gene candidates that are co-linear with the rat U1-RNA, 3.6kb apart and in the opposite orientation. The two genes are surrounded by identical sequences of 491bp upstream and 178bp downstream. The upstream sequences do not contain a TATA box, but share many block homologies with those for the human U1-RNA gene(1-3). A 101bp "identifier (ID) sequence", which was reported to be specifically expressed in rat brain (4), is inserted immediately after the shared sequence downstream of one of the genes. In the fourth clone, there are two putative pseudogenes, which have one or three nucleotide changes, 3kb apart and in the same orientation. Southern blot analysis of total rat DNA reveals about 50 U1-RNA genes/pseudogenes in the genome.     

Three types of rat U1 small nuclear RNA genes with different flanking sequences are induced to express in vivo

There are about 50 copies of U1 RNA genes/pseudogenes in the rat genome. To date, we have isolated so far 25 phage clones carrying a U1 RNA gene/pseudogene from two rat genomic libraries. The 12 clones were selected by hybridization with the U1 RNA coding region under a stringent condition, and were mapped and sequenced. Here, we report three types of U1 RNA genes with different flanking sequences, all of which were shown to be induced to express in vivo by transfection with their polylinker-inserted maxi U1 RNA genes into cultured rat cells. Although these three classes of U1 RNA genes have few homologous flanking sequences, they provide both upstream and downstream of the genes two conserved blocks, which may possibly play an important role in U1 RNA expression.     

Human DNA sequences complementary to the small nuclear RNA U2.

Clones containing sequences complementary to the small nuclear RNA U2 were isolated from a human DNA library (1). Three clones, designated U2/4, U2/6 and U2/7 were purified and characterized by restriction enzyme cleavage, hybridization and heteroduplex analysis. Hybridization showed that the three clones each contained one single region which is complementary to U2 RNA. Restriction enzyme cleavage revealed furthermore that the inserted fragments in the three recombinants are different. Heteroduplex analysis identified a 240-380 bp long duplex region in each heteroduplex which includes sequences complementary to U2 RNA. Heteroduplexes between clones U2/4 and U2/7 as well as between U2/4 and U2/6 revealed two additional approximately 200 bp long homologies. The remainder of the inserts were found to lack measurable sequence homology. Two fragments from clone U2/4 were subcloned in the pBR322 vector and the subclones were used to determine the nucleotide sequence of a region in clone U2/4 which is complementary to U2 RNA. A comparison between the established sequence and the sequence for rat U2 RNA (2) reveals several discrepancies.     

A U6 snRNA gene with an internal promoter is juxtaposed to an snRNP protein sequence within an intron of a human G protein gene.

A complex locus on human chromosome 1 brings together sequences homologous to a G protein and two components of the RNA processing machinery of eukaryotic cells. Specifically, the seventh intron of the human Gi3 alpha gene contains a fusion of a partial snRNP E protein pseudogene to a variant U6 snRNA gene. The novel U6 sequence contains nine point mutations and a one nucleotide deletion relative to the major U6 genes from humans. Unlike all other vertebrate U6 genes characterized to date, the variant U6 gene is efficiently transcribed by RNA polymerase III even in the absence of all natural flanking sequences. The union of elements from the signal transduction pathway and the RNA processing machinery suggests the possibility of functional interplay.     

Drosophila melanogaster genes for U1 snRNA variants and their expression during development.

We have cloned and characterized a complete set of seven U1-related sequences from Drosophila melanogaster. These sequences are located at the three cytogenetic loci 21D, 82E, and 95C. Three of these sequences have been previously studied: one U1 gene at 21D which encodes the prototype U1 sequence (U1a), one U1 gene at 82E which encodes a U1 variant with a single nucleotide substitution (U1b), and a pseudogene at 82E. The four previously uncharacterized genes are another U1b gene at 82E, two additional U1a genes at 95C, and a U1 gene at 95C which encodes a new variant (U1c) with a distinct single nucleotide change relative to U1a. Three blocks of 5' flanking sequence similarity are common to all six full length genes. Using specific primer extension assays, we have observed that the U1b RNA is expressed in Drosophila Kc cells and is associated with snRNP proteins, suggesting that the U1b-containing snRNP particles are able to participate in the process of pre-mRNA splicing. We have also examined the expression throughout Drosophila development of the two U1 variants relative to the prototype sequence. The U1c variant is undetectable by our methods, while the U1b variant exhibits a primarily embryonic pattern reminiscent of the expression of certain U1 variants in sea urchin, Xenopus, and mouse.     

Linkage map of two HLA-SB beta and two HLA-SB alpha-related genes: an intron in one of the SB beta genes contains a processed pseudogene

Three overlapping cosmid clones contain coding sequences for four HLA Class II genes, provisionally identified as two HLA-SB alpha and two HLA-SB beta genes. The genes are in the order beta, alpha, beta, alpha, inverted with respect to each other. One of the SB beta genes contains a 513 bp sequence that appears to be a processed pseudogene, flanked by direct 17 bp repeat sequences, in the intron upstream of the beta 1 exon. The pseudogene is homologous to a family of sequences of approximately 25-40 members, most of which are not on chromosome 6. A cDNA clone, highly homologous to the pseudogene, except for its 5' end, contains a normal poly(A) addition site and a poly(A) tail. The cDNA clone is homologous to a single-copy gene in both man and mouse, encoded on human chromosome 15. A search of published DNA sequences identified a mouse sequence, with about 77% similarity to the pseudogene sequence, in the negative strand of an intron in a mouse dihydrofolate reductase gene. The second SB beta gene does not contain the pseudogene sequence.     

The cDNA sequences of the sea urchin U7 small nuclear RNA suggest specific contacts between histone mRNA precursor and U7 RNA during RNA processing.

3' Processing of sea urchin H3 histone pre-mRNA depends on a small nuclear RNP which contains an RNA of nominally 60 nucleotide length, referred to below as U7 RNA. The U7 RNA can be enriched by precipitation of sea urchin U-snRNPs with human systematic lupus erythematosus antiserum of the Sm serotype. We have prepared cDNA clones of U7 RNA and determined by hybridization techniques that this RNA is present in sea urchin eggs at 30-fold lower molar concentration than U1 RNA. The RNA sequences derived from an analysis of eight U7 cDNA clones show neither homologies nor complementarities to any other know U-RNAs. The 3' portion of the presumptive RNA sequence can be folded into a stem-loop structure. The 5'-terminal sequences would be largely unstructured as free RNA. Their most striking feature is their base complementarity to the 3' conserved sequences of histone pre-mRNAs. Six out of nine bases of the conserved CAAGAAAGA sequence of the histone mRNA precursor and 13 out of 16 nucleotides from the conserved palindrome can be base paired with presumptive U7 RNA sequence, suggesting a unique hybrid structure for a processing intermediate formed from histone precursor and U7 RNA.     

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 cytoskeletal gamma-actin: analysis and implications of the structure of cloned cDNA and processed pseudogenes

The nucleotide sequence corresponding to almost the whole of a mouse gamma-cytoskeletal actin mRNA was determined from overlapping cloned DNA copies derived from brain mRNA. Several gamma-actin processed pseudogenes were isolated from a library of cloned DBA mouse genomic DNA, and the nucleotide sequences of these were determined and compared with that of the cDNA. This showed that two of these pseudogenes had arisen from a gene duplication or amplification event, and indicated that they had subsequently undergone partial correction against one another. The relative ages of the pseudogenes were estimated on the basis of their percentage divergence from the cDNA sequence and these were compared with an estimation based on the number of presumed silent mutations in the cDNA since each pseudogene had arisen. Consistent results were obtained, except in the case of one pseudogene which also showed an anomalous regional distribution of differences from the cDNA sequence. One way of accounting for the features of this anomalous pseudogene is by postulating that it is derived from a second functional gene for gamma-actin, different from that represented by the cDNA described here.     

Structure of an unusual sea urchin U1 RNA gene cluster

Genomic clones containing multiple copies of the Lytechinus variegatus U1 gene have been isolated from a gene library in the phage lambda EMBL3. These clones contain both types of U1 RNA gene repeats interspersed in the same 15-kb fragment. In addition, about 1/3 of the repeat units contain a 260-bp insert 460 bp prior to the first nucleotide of the U1 RNA sequence. The inserted sequence is abundant in the sea urchin genome as judged by Southern blots of genomic DNA. There are no repeated sequences flanking the insert. The insert occurs at the same position in the highly conserved 5'-flanking region at which a deletion has previously been reported.     

Evolution of human cytoplasmic actin gene sequences: chromosome mapping and structural characterizations of three cytoplasmic actin-like pseudogenes including one on the Y chromosome

Eight recombinant phage clones containing cytoplasmic actin-like gene sequences have been isolated from a human genomic library for structural characterization. Kpn I family repeat sequences flank six of these actin genes isolated, and Alu family repeats are scattered throughout the DNA inserts of all eight phage clones. Three of these genes are γ actin-like, and the other five are β actin-like. The complete nucleotide sequence analysis of one β and one γ actin-like genes and their flanking regions demonstrates that they both are processed pseudogenes. Using unique DNA sequences flanking these two pseudogenes as hybridization probes for human-mouse somatic cell hybrid DNAs, we have mapped the two actin pseudogenes on human chromosomes 8 and 3, respectively. We have also determined the DNA sequence of a human Y chromosome-linked, processed actin pseudogene. The different values of sequence divergence of these processed pseudogenes and their functional counterparts allow us to estimate the time of generation of the pseudogenes. The results suggest that the cDNA insertion events generating the human cytoplasmic actin-like pseudogenes have occurred at significantly different times during the evolution of primates, after their separation from other mammalian species.     

Nonrandom integration of human U4 RNA pseudogenes.

Four loci for human U4 RNA have been characterized by DNA sequence analysis. The results show that all four loci represent pseudogenes, which are flanked by direct repeats. Three of the pseudogenes, designated U4/5, U4/6, and U4/8, have very similar structures; they are all truncated and contain the first 67 to 68 nucleotides of the U4 RNA sequence. Their properties suggest that they were created by integration of truncated cDNA copies of the U4 RNA into new chromosomal sites. An interesting observation was that their flanking regions exhibit sequence homology. A purine-rich 5'-flanking sequence 12 to 13 nucleotides long is almost perfectly conserved in all three loci. Boxes of homology were also found on the 3' side when the U4/6 and U4/8 loci were compared. The U4/4 locus has a slightly different structure; the pseudogene matches the first 79 nucleotides of U4 RNA, but contains a greater number of mutations than the other pseudogenes. Taken together, the results suggest that a frequently occurring type of pseudogene for human U4 was created by a RNA-mediated mechanism and that the integration sites have features in common.