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).     

Structures of four human pseudogenes for U7 small nuclear RNA

Four U7 RNA-related sequences were isolated from a human genomic DNA library. None of the sequences completely match the published human U7 RNA sequence and all of them contain features typical of reverse-transcribed pseudogenes.     

Drosophila melanogaster U1 and U2 small nuclear RNA genes contain common flanking sequences

The flanking sequences of three U2 genes (or pseudogenes) and one U1 gene of Drosophila melanogaster have been determined. Comparison of the sequences reveals a remarkable homology between position ?30 and ?65 upstream from the structural genes, starting with a TATA box-like sequence. The 3′ flanking regions are also conserved in all genes and contain a canonical A-A-T-A-A-A polyadenylation signal.     

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.     

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.     

Isolation of an active gene and of two pseudogenes for mouse U7 small nuclear RNA

Three U7 RNA-related sequences were isolated from mouse genomic DNA libraries. Only one of the sequences completely matches the published mouse U7 RNA sequence, whereas the other two apparently represent pseudogenes. The matching sequence represents a functional gene, as it is expressed after microinjection into Xenopus laevis oocytes. Sequence variations of the conserved cis-acting 5' and 3' elements of U RNA genes may partly explain the low abundance of U7 RNA.     

Human U1 small nuclear RNA pseudogenes do not map to the site of the U1 genes in 1p36 but are clustered in 1q12-q22.

Human U1 small nuclear RNA is encoded by approximately 30 gene copies. All of the U1 genes share several kilobases of essentially perfect flanking homology both upstream and downstream from the U1 coding region, but remarkably, for many U1 genes excellent flanking homology extends at least 24 kilobases upstream and 20 kilobases downstream. Class I U1 RNA pseudogenes are abundant in the human genome. These pseudogenes contain a complete but imperfect U1 coding region and possess extensive flanking homology to the true U1 genes. We mapped four class I pseudogenes by in situ hybridization to the long arm of chromosome 1, bands q12-q22, a region distinct from the site on the distal short arm of chromosome 1 to which the U1 genes have been previously mapped (Lund et al., Mol. Cell. Biol. 3:2211-2220, 1983; Naylor et al., Somat. Cell Mol. Genet. 10:307-313, 1984). We confirmed our in situ hybridization results by genomic blotting experiments with somatic cell hybrid lines with translocation products of human chromosome 1. These experiments provide further evidence that class I U1 pseudogenes and the true U1 genes are not interspersed. The results, along with those published elsewhere (Bernstein et al., Mol. Cell. Biol. 5:2159-2171, 1985), suggest that gene amplification may be responsible for the sequence homogeneity of the human U1 gene family.     

Loop I of U1 small nuclear RNA is the only essential RNA sequence for binding of specific U1 small nuclear ribonucleoprotein particle proteins.

The binding of the U1 small nuclear ribonucleoprotein (snRNP)-specific proteins C, A, and 70K to U1 small nuclear RNA (snRNA) was analyzed. Assembly of U1 snRNAs from bean and soybean and a set of mutant Xenopus U1 snRNAs into U1 snRNPs in Xenopus egg extracts was studied. The ability to bind proteins was analyzed by immunoprecipitation with monospecific antibodies and by a protein-sequestering assay. The only sequence essential for binding of the U1-specific proteins was the conserved loop sequence in the 5' hairpin of U1. Further analysis suggested that protein C binds directly to the loop and that the assembly of proteins A and 70K into the RNP requires mainly protein-protein interactions. Protein C apparently recognizes a specific RNA sequence rather than a secondary structural element in the RNA.     

Precursors of U4 small nuclear RNA

The processing and ribonucleoprotein assembly of U4 small nuclear RNA has been investigated in HeLa cells. After a 45-min pulse label with [3H]uridine, a set of apparently cytoplasmic RNAs was observed migrating just behind the gel electrophoretic position of mature U4 RNA. These molecules were estimated to be one to at least seven nucleotides longer than mature U4 RNA. They reacted with Sm autoimmune patient sera and a monoclonal Sm antibody, indicating their association with proteins characteristic of small nuclear ribonucleoprotein complexes. The same set of RNAs was identified by hybrid selection of pulse-labeled RNA with cloned U4 DNA, confirming that these are U4 RNA sequences. No larger nuclear precursors of these RNAs were detected. Pulse-chase experiments revealed a progressive decrease in the radioactivity of the U4 precursor RNAs coincident with an accumulation of labeled mature U4 RNA, confirming a precursor-product relationship.     

Human U1 small nuclear RNA genes: extensive conservation of flanking sequences suggests cycles of gene amplification and transposition.

The DNA immediately flanking the 164-base-pair U1 RNA coding region is highly conserved among the approximately 30 human U1 genes. The U1 multigene family also contains many U1 pseudogenes (designated class I) with striking although imperfect flanking homology to the true U1 genes. Using cosmid vectors, we now have cloned, characterized, and partially sequenced three 35-kilobase (kb) regions of the human genome spanning U1 homologies. Two clones contain one true U1 gene each, and the third bears two class I pseudogenes 9 kb apart in the opposite orientation. We show by genomic blotting and by direct DNA sequence determination that the conserved sequences surrounding U1 genes are much more extensive than previously estimated: nearly perfect sequence homology between many true U1 genes extends for at least 24 kb upstream and at least 20 kb downstream from the U1 coding region. In addition, the sequences of the two new pseudogenes provide evidence that class I U1 pseudogenes are more closely related to each other than to true genes. Finally, it is demonstrated elsewhere (Lindgren et al., Mol. Cell. Biol. 5:2190-2196, 1985) that both true U1 genes and class I U1 pseudogenes map to chromosome 1, but in separate clusters located far apart on opposite sides of the centromere. Taken together, these results suggest a model for the evolution of the U1 multigene family. We speculate that the contemporary family of true U1 genes was derived from a more ancient family of U1 genes (now class I U1 pseudogenes) by gene amplification and transposition. Gene amplification provides the simplest explanation for the clustering of both U1 genes and class I pseudogenes and for the conservation of at least 44 kb of DNA flanking the U1 coding region in a large fraction of the 30 true U1 genes.     

Identification of molecular contacts between the U1 A small nuclear ribonucleoprotein and U1 RNA.

We recently determined the crystal structure of the RNP domain of the U1 small nuclear ribonucleoprotein A and identified Arg and Lys residues involved in U1 RNA binding. These residues are clustered around the two highly conserved segments, RNP1 and RNP2, located in the central two beta strands. We have now studied the U1 RNA binding of mutants where potentially hydrogen bonding residues on the RNA binding surface were replaced by non-hydrogen bonding residues. In the RNP2 segment, the Thr11----Val and Asn15----Val mutations completely abolished, and the Tyr13----Phe and Asn16----Val mutations substantially reduced the U1 RNA binding, suggesting that these residues form hydrogen bonds with the RNA. In the RNP1 segment Arg52----Gln abolished, but Arg52----Lys only slightly affected U1 RNA binding, suggesting that Arg52 may form a salt bridge with phosphates of U1 RNA. Ethylation protection experiments of U1 RNA show that the backbone phosphates of the 3' two-thirds of loop II and the 5' stem are in contact with the U1 A protein. The U1 A protein-U1 RNA binding constant is substantially reduced by A----G and G----A replacements in loop II, but not by C----U or U----C replacements. Based on these biochemical data we propose a structure for the complex between the U1 A ribonucleoprotein and U1 RNA.     

Architecture of the U5 small nuclear RNA.

We have used comparative sequence analysis and deletion analysis to examine the secondary structure of the U5 small nuclear RNA (snRNA), an essential component of the pre-mRNA splicing apparatus. The secondary structure of Saccharomyces cerevisiae U5 snRNA was studied in detail, while sequences from six other fungal species were included in the phylogenetic analysis. Our results indicate that fungal U5 snRNAs, like their counterparts from other taxa, can be folded into a secondary structure characterized by a highly conserved stem-loop (stem-loop 1) that is flanked by a moderately conserved internal loop (internal loop 1). In addition, several of the fungal U5 snRNAs include a novel stem-loop structure (ca. 30 nucleotides) that is adjacent to stem-loop 1. By deletion analysis of the S. cerevisiae snRNA, we have demonstrated that the minimal U5 snRNA that can complement the lethal phenotype of a U5 gene disruption consists of (i) stem-loop 1, (ii) internal loop 1, (iii) a stem-closing internal loop 1, and (iv) the conserved Sm protein binding site. Remarkably, all essential, U5-specific primary sequence elements are encoded by a 39-nucleotide domain consisting of stem-loop 1 and internal loop 1. This domain must, therefore, contain all U5-specific sequences that are essential for splicing activity, including binding sites for U5-specific proteins.     

A candidate U1 small nuclear RNA for trypanosomatid protozoa.

In trypanosomatid protozoa, all mRNAs obtain identical 5'-ends by trans-splicing of the 5'-terminal 39 nucleotides of a small spliced leader RNA to appropriate acceptor sites in pre-mRNA. Although this process involves spliceosomal small nuclear (sn) RNAs, it is thought that trypanosomatids do not contain a homolog of the cis-spliceosomal U1 snRNA. We show here that a trypanosomatid protozoon, Crithidia fasciculata, contains a novel small RNA that displays several features characteristic of a U1 snRNA, including (i) a methylguanosine cap and additional 5'-terminal modifications, (ii) a potential binding site for common core proteins that are present in other trans-spliceosomal ribonucleoproteins, (iii) a U1-like 5'-terminal sequence, and (iv) a U1-like stem/loop I structure. Because trypanosomatid pre-mRNAs do not appear to contain cis-spliced introns, we argue that this previously unrecognized RNA species is a good candidate to be a trans-spliceosomal U1 snRNA.     

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.