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.     

Divergence of U2 snRNA sequences in the genome of D. melanogaster.

Four different U2-snRNA genes/related sequences of D. melanogaster were cloned and characterized. The sequences of all four genes suggest that they were generated by a DNA-mediated mechanism. These genes/related sequences were found to be located in two loci, each locus containing two U2 snRNA sequences. Using coding sequences as well as flanking sequences as hybridization probes against polytene chromosomes of D. melanogaster Oregon R we were able to map these loci separately at positions 34BC and 84C. By Northern analysis we observed that the quantities of U2- and U1-snRNA are coordinated and change during the embryonic development of the fly.     

Control of mouse U1a and U1b snRNA gene expression by differential transcription.

The expression of mouse embryonic U1 snRNA (mU1b) genes is subject to stage- and tissue-specific control, being restricted to early embryos and adult tissues that contain a high proportion of stem cells capable of further differentiation. To determine the mechanism of this control we have sought to distinguish between differential RNA stability and regulation of U1 gene promoter activity in several cell types. We demonstrate here that mU1b RNA can accumulate to high levels in permanently transfected mouse 3T3 and C127 fibroblast cells which normally do not express the endogenous U1b genes, and apparently can do so without significantly interfering with cell growth. Expression of transfected chimeric U1 genes in such cells is much more efficient when their promoters are derived from a constitutively expressed mU1a gene rather than from an mU1b gene. In transgenic mice, introduced U1 transgenes with an mU1b 5' flanking region are subject to normal tissue-specific control, indicating that U1b promoter activity is restricted to tissues that normally express U1b genes. Inactivation of the embryonic genes during normal differentiation is not associated with methylation of upstream CpG-rich sequences; however, in NIH 3T3 fibroblasts, the 5' flanking regions of endogenous mU1b genes are completely methylated, indicating that DNA methylation serves to imprint the inactive state of the mU1b genes in cultured cells. Based on these results, we propose that the developmental control of U1b gene expression is due to differential activity of mU1a and mU1b promoters rather than to differential stability of U1a and U1b RNAs.     

Human U1 RNA pseudogenes may be generated by both DNA- and RNA-mediated mechanisms.

Analysis of cloned human genomic loci homologous to the small nuclear RNA U1 established that such sequences are abundant and dispersed in the human genome and that only a fraction represent bona fide genes. The majority of genomic loci bear defective gene copies, or pseudogenes, which contain scattered base mismatches and in some cases lack the sequence corresponding to the 3' end of U1 RNA. Although all of the U1 genes examined to date are flanked by essentially identical sequences and therefore appear to comprise a single multigene family, we present evidence for the existence of at least three structurally distinct classes of U1 pseudogenes. Class I pseudogenes had considerable flanking sequence homology with the U1 gene family and were probably derived from it by a DNA-mediated event such as gene duplication. In contrast, the U1 sequence in class II and III U1 pseudogenes was flanked by single-copy genomic sequences completely unrelated to those flanking the U1 gene family; in addition, short direct repeats flanked the class III but not the class II pseudogenes. We therefore propose that both class II and III U1 pseudogenes were generated by an RNA-mediated mechanism involving the insertion of U1 sequence information into a new chromosomal locus. We also noted that two other types of repetitive DNA sequences in eucaryotes, the Alu family in vertebrates and the ribosomal DNA insertions in Drosophila, bore a striking structural resemblance to the classes of U1 pseudogenes described here and may have been created by an RNA-mediated insertion event.     

Cloning and characterization of two processed pseudogenes and the cDNA for the murine U1 snRNP-specific protein C

Genes for the snRNP proteins U1-70K, U1-A, Sm-B′/B, Sm-D1 and Sm-E have been isolated from various metazoan species. The genes for Sm-D1 and Sm-E, which were isolated from a murine and human source respectively, appear to belong to a multigene family. It has been suggested that also for the mammalian U1-C protein such a multigene family exists. With the human U1-C cDNA as a probe, two genes containing sequences homologous to the probe sequence were isolated from a mouse genomic library. Simultaneously, a murine U1-C cDNA was isolated from a mouse cDNA library. This 0.74 kb cDNA contains an open reading frame (ORF) of 477 bp encoding a polypeptide of 159 amino acids (aa) which differs at only one position (position 65) from the human U1-C protein. One of the isolated U1-C genes contains an ORF as well and shares 92% nucleotide sequence identity with the mouse U1-C cDNA. The features of this gene, in particular the absence of introns, the acquisition of a 3′ poly(A) tail and flanking direct repeats, indicate that it represents a processed pseudogene. At the predicted aa sequence level, substitutions of conserved residues at functionally important positions are observed, strongly suggesting that expression of this gene would not lead to a functional polypeptide. The second U1-C gene appeared to be a pseudogene as well because it is also intronless and contains a frameshift mutation compared to the ORF in the mouse U1-C cDNA. The characterization of these two pseudogenes points to the existence of a U1-C multigene family in mice. Furthermore, comparison of aa sequences of the murine, human and Xenopus U1-C shows that the protein is highly conserved through evolution. Since the Xenopus U1-C differs from the two mammalian counterparts solely at a number of positions in the C-terminal region, it can be concluded that aa changes are less well tolerated in the N-terminal region of U1-C than in the rest of the protein.     

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.     

Using Profiles Based on Nucleotide Hydrophobicity to Define Essential Regions for Splicing

The splice-site sequences of U2-type introns are highly degenerate, so many different sequences can function as U2-type splice sites. Using our new profiles based on hydrophobicity properties we pointed out specific properties for regions surrounding splice sites. We built a set T of flanking regions of genes with 1-3 introns from 21st and 22nd chromosomes extracted from GenBank to define positions having conserved properties, namely hydrophobicity, that are potentially essential for recognition by spliceosome.     

A complete and a truncated U1 snRNA gene of Drosophila melanogaster are found as inverted repeats at region 82E of the polytene chromosomes.

A phage containing two sequences homologous to U1 snRNA was isolated from a Drosophila melanogaster genomic library, and identified with a previously cloned D. melanogaster U1 snRNA gene. DNA sequence analysis showed that complete and truncated U1 snRNA genes are present, both of which have base substitutions relative to U1 snRNA. These genes show conservation of 5' and 3' flanking regions relative to other U1 and U2 snRNA genes of Drosophila. Intramolecular renaturation experiments and electron microscope mapping demonstrates that the two U1 snRNA sequences are present as inverted repeats about 2.7kb apart, separated by a smaller pair of inverted repeats of an unrelated sequence. These U1 snRNA sequences were located by in situ hybridization at 82E, and related sequences were found at 21D and 95C on the polytene chromosome map. The results are discussed with reference to the origin and function of snRNAs.     

Determinants of plant U12-dependent intron splicing efficiency

Factors affecting splicing of plant U12-dependent introns have been examined by extensive mutational analyses in an in vivo tobacco (Nicotiana tabacum) protoplast system using introns from three different Arabidopsis thaliana genes: CBP20, GSH2, and LD. The results provide evidence that splicing efficiency of plant U12 introns depends on a combination of factors, including UA content, exon bridging interactions between the U12 intron and flanking U2-dependent introns, and exon splicing enhancer sequences (ESEs). Unexpectedly, all three plant U12 introns required an adenosine at the upstream purine position in the branchpoint consensus UCCUURAUY. The exon upstream of the LD U12 intron is a major determinant of its higher level of splicing efficiency and potentially contains two ESE regions. These results suggest that in plants, U12 introns represent a level at which expression of their host genes can be regulated.