U2 and U1 snRNA gene loci associate with coiled bodies

The coiled bodies are nuclear structures rich in a variety of nuclear and nucleolar components including snRNAs. We have investigated the possibility that coiled bodies may associate with snRNA genes and report here that there is a high degree of association between U2 and U1 genes with a subset of coiled bodies. As investigated in human HeLa cells grown in monolayer culture, about 75% of the nuclei had at least one U2 gene associated with a coiled body, and 45% had at least one U1 locus associated. In another suspension-grown HeLa cell strain, 92% of cells showed association of one or more U2 genes with coiled bodies. In contrast to the U2 and U1 gene associations, a locus closely linked to the U2 gene cluster appeared associated with a coiled body only in 10% of cells. Associated snRNA gene signals were repeatedly positioned at the edge of the coiled body. Thus, this association was highly nonrandom and spatically precise. Our analysis revealed a much higher frequency of association for closely spaced “doublet” U2 gene signals, with over 80% of paired signals associated as opposed to 35% for single U2 signals. This finding, coupled with the fact that not all genes were associated in all cells, suggested the possibility of a cell-cycle-dependent, possibly S-phase, association. However, an analysis of S- and non-S-phase cells using BrdU incorporation or cell synchronization did not indicate an increased level of association in S-phase. These and other results suggested that a substantial fraction of paired U2 signals represented association of U2 genes on homologous chromosomes rather than only replicated DNA. Furthermore, triple lable analysis showed that in a significant fraction of cells U1 and U2 genes were both associated with the same coiled body. U1 and U2 genes were closely paired in approximately 20% of cells, over 60% of which were associated with a readily identifiable coiled body. This finding raises the possibility that multiple genes of a particular class may be in association with each coiled body. Thus, the coiled body may be a dynamic structure which transiently interacts with or is formed by one or more specific genetic loci, possibly carrying out some function related to their expression. © 1995 Wiley-Liss, Inc.     

Novel structure of a human U6 snRNA pseudogene

A genomic DNA library containing human placental DNA cloned into phage lambda Charon 4A was screened for snRNA U6 genes. In vitro 32P-labeled U6 snRNA isolated from HeLa cells was used as a hybridization probe. A positive clone containing a 4.6-kb EcoRI fragment of human chromosomal DNA was recloned into the EcoRI site of pBR325 and mapped by restriction endonuclease digestion. Restriction fragments containing U6 RNA sequences were identified by hybridization with isolated U6[32P]RNA. The sequence analysis revealed a novel structure of a U6 RNA pseudogene, bearing two 17-nucleotide(nt)-long direct repeats of genuine U6 RNA sequences arranged in a head-to-tail fashion within the 5' part of the molecule. Hypothetical models as to how this type of snRNA U6 pseudogene might have been generated during evolution of the human genome are presented. When compared to mammalian U6 RNA sequences the pseudogene accounts for a 77% overall sequence homology and contains the authentic 5'- and 3'-ends of the U6 RNA.     

Human U2 snRNA genes exhibit a persistently open transcriptional state and promoter disassembly at metaphase

In mammals, small multigene families generate spliceosomal U snRNAs that are nearly as abundant as rRNA. Using the tandemly repeated human U2 genes as a model, we show by footprinting with DNase I and permanganate that nearly all sequences between the enhancer-like distal sequence element and the initiation site are protected during interphase whereas the upstream half of the U2 snRNA coding region is exposed. We also show by chromatin immunoprecipitation that the SNAPc complex, which binds the TATA-like proximal sequence element, is removed at metaphase but remains bound under conditions that induce locus-specific metaphase fragility of the U2 genes, such as loss of CSB, BRCA1, or BRCA2 function, treatment with actinomycin D, or overexpression of the tetrameric p53 C terminus. We propose that the U2 snRNA promoter establishes a persistently open state to facilitate rapid reinitiation and perhaps also to bypass TFIIH-dependent promoter melting; this open state would then be disassembled to allow metaphase chromatin condensation.     

A UV-crosslinkable interaction in human U6 snRNA.

U6 snRNA is the most conserved of all the snRNAs involved in pre-mRNA splicing, and likely plays an important role in splicing catalysis. Using a U6 snRNA fragment encompassing residues 25-99, we have identified a strong, UV-sensitive tertiary intramolecular interaction. A 5' deletion that removed sequences up to nt 37 only slightly reduced crosslinking, but further deletion of 11 bases, eliminating the nearly invariant ACAGAGA sequence, essentially abolished crosslinking, as did deletion of sequences 3' of 82A. The crosslinked residues were mapped to 44G in the ACAGAGA sequence and to 81C, the nucleotide at the base of the U6 intramolecular helix, opposite the G of the invariant AGC trinucleotide. This interaction is striking in that it has the potential to juxtapose invariant regions of U6 believed to play critical roles in splicing catalysis.     

Structure and expression of the U5 snRNA gene of Arabidopsis thaliana. Conserved upstream sequence elements in plant U-RNA genes.

We have previously characterized the U2 small nuclear (sn) RNA gene family of Arabidopsis thaliana. To find out the structural features of upstream and downstream non-coding regions that are shared by different U-RNA genes in higher plants we have isolated the gene encoding a 125 nt-long U5 snRNA of Arabidopsis. Activity of the cloned gene was demonstrated in stably transformed tobacco calli and by transient expression in transfected protoplasts of Nicotiana plumbaginifolia. Southern analysis indicated that the Arabidopsis genome contains 8-9 copies of the U5 gene. Alignment of upstream non-coding regions revealed two elements conserved between all plant U-RNA genes characterized so far: the sequence RTCCCACATCG (-70/-80 region, 100% conservation) and the TATA homology around position -30. The coding regions in all genes are followed by the sequence CAN4-9AGTN (A/T)AA which may correspond to a termination and/or processing signal.     

Amplification of plant U3 and U6 snRNA gene sequences using primers specific for an upstream promoter element and conserved intragenic regions.

U-snRNA genes in higher plants contain two essential promoter elements, the USE with sequence RTCCCACATCG and the TATA-like box, positioned in the -70 and -30 regions, respectively. Using an oligodeoxynucleotide containing the USE motif and oligodeoxynucleotides specific for the intragenic regions conserved in U-snRNAs, several sequences encoding U6 and U3 snRNAs were determined by polymerase chain reaction (PCR) amplification of Arabidopsis thaliana and tobacco genomic DNAs. This method provides a simple and rapid procedure for characterisation of plant U-snRNA genes and their promoters. It could also be used for the characterisation of other genes containing conserved upstream promoter elements. PCR-derived fragments were used as probes for the isolation of the U3 snRNA genes from a genomic library of Arabidopsis. Two isolated U3 genes were shown to be active when transfected into protoplasts of Nicotiana plumbaginifolia. Both U3 genes contain the USE and TATA-like upstream elements located in similar positions to the U6 genes of Arabidopsis. The encoded Arabidopsis U3 snRNAs can be folded into a secondary structure which is more similar to that of U3 RNAs from lower eukaryotes rather than from metazoa.