Limited functional equivalence of phylogenetic variation in small nuclear RNA: yeast U2 RNA with altered branchpoint complementarity inhibits splicing and produces a dominant lethal phenotype.

U2 is a highly conserved small nuclear RNA essential for pre-mRNA splicing in mammals and yeast and for trans-splicing in trypanosomes. To test the function of variant U2 RNA structures from different organisms, we conducted phylogenetic exchanges of U2 domains. Replacing nucleotides 1-120 of yeast U2 with the corresponding region of human U2 generates a U2 RNA that is correctly folded and functions in yeast.

Lethal and temperature-sensitive mutations and their suppressors identify an essential structural element in U2 small nuclear RNA.

U2 snRNA is an essential component of the splicing apparatus in eukaryotic cells. Three possible secondary structures for the highly conserved 5' half of U2 snRNA are consistent with U2 phylogenetic sequence variation. To distinguish among these models and to test the function of U2 structural elements, we made greater than 35 mutations in the yeast U2 snRNA gene.

Internal sequences that distinguish yeast from metazoan U2 snRNA are unnecessary for pre-mRNA splicing.

U2 small nuclear RNA is a highly conserved component of the eukaryotic cell nucleus involved in splicing messenger RNA precursors. In the yeast Saccharomyces cerevisiae, U2 RNA interacts with the intron by RNA-RNA pairing between the conserved branchpoint sequence UACUAAC and conserved nucleotides near the 5' end of U2 (ref. 4).

Distinct factors with Sp1 and NF-A specificities bind to adjacent functional elements of the human U2 snRNA gene enhancer.

The enhancer regions of mammalian and avian U1 and U2 small nuclear RNA (snRNA) genes are unusual in containing the sequence GGGCGG (GC-box) immediately upstream from the sequence ATGCAAAT (octamer). We made point mutations in the human U2 snRNA enhancer and tested them for the ability to direct U2 transcription in HeLa cells, as well as for the ability to form complexes with factors present in HeLa cell nuclear extracts. We show that neither the GC-box nor the octamer alone is sufficient for enhancer activity in vivo.

U2 RNA from yeast is unexpectedly large and contains homology to vertebrate U4, U5, and U6 small nuclear RNAs.

I have determined the structure of the gene from Saccharomyces cerevisiae coding for the yeast homolog of vertebrate U2 snRNA. Surprisingly, the RNA is 1175 nucleotides long, six times larger than U2 RNAs from other organisms, including Schizosaccharomyces pombe. Nearly 100 nucleotides of the large RNA share sequence homology and potential secondary structure with metazoan U2.

Human U2 small nuclear RNA genes contain an upstream enhancer.

The human U1 and U2 snRNA genes lack an obvious TATA box, but are extremely powerful RNA polymerase II transcription units capable of accurately initiating at least one transcript per gene every 2-4 s. We have investigated the location of cis-acting regulatory elements within the flanking sequences of human U2 and U1 genes. By introducing marked human U2 genes into HeLa cells on SV40- and pUC13-based vectors, we found that transient expression of the marked U2 gene did not require the SV40 enhancer.

Sequences required for 3' end formation of human U2 small nuclear RNA.

Xenopus oocytes injected with human U2 snRNA genes synthesize mature U2 as well as a U2 precursor with about 10 extra 3' nucleotides (human pre-U2 RNA). Formation of the pre-U2 3' end requires a downstream element located between position +16 and +37 in the U2 3'-flanking sequence. The distance between this element and the U2 coding region can be increased without affecting formation of the pre-U2 3' end.

U1 small nuclear RNA genes are subject to dosage compensation in mouse cells.

Multiple copies of a gene that encodes human U1 small nuclear RNA were introduced into mouse C127 cells with bovine papilloma virus as the vector. For some recombinant constructions, the human U1 gene copies were maintained extrachromosomally on the viral episome in an unrearranged fashion. The relative abundance of human and mouse U1 small nuclear RNA varied from one cell line to another, but in some lines human U1 RNA accounted for as much as one-third of the total U1.

Orientation-dependent transcriptional activator upstream of a human U2 snRNA gene.

We examined the structure of the promoter for the human U2 snRNA gene, a strong RNA polymerase II transcription unit without an obvious TATA box. A set of 5' deletions was constructed and assayed for the ability to direct initiation of U2 snRNA after microinjection into Xenopus oocytes. Sequences between positions -295 and -218 contain an activator element which stimulates accurate initiation by 20- to 50-fold, although as few as 62 base pairs of 5' flanking sequence are sufficient to direct the accurate initiation of U2 RNA.

Human genes for U2 small nuclear RNA map to a major adenovirus 12 modification site on chromosome 17.

U2 RNA is one of the abundant, highly conserved species of small nuclear RNA (snRNA) molecules implicated in RNA processing. As is typical of mammalian snRNAs, human U1 and U2 are each encoded by a multigene family. In the human genome, defective copies of the genes (pseudogenes) far outnumber the authentic genes.

Analysis of transcription during the cell cycle in toluenized Chlamydomonas reinhardi cells.

A toluene-permeabilized cell system was established to examine the transcription of certain RNAs regulated during the cell cycle in Chlamydomonas reinhardi. The incorporation of [alpha-32P]UTP into RNA which hybridizes to specific cloned cDNA, such as beta-tubulin, indicates that the cell cycle pattern of RNA accumulation may be controlled, in part, by differential transcription.

Cell cycle stage-specific accumulation of mRNAs encoding tubulin and other polypeptides in Chlamydomonas.

The accumulation pattern of a number of mRNAs during the cell cycle of Chlamydomonas was examined by two-dimensional gel analysis of in vitro translation products and by RNA blot hybridization analysis. Two-dimensional gel analysis revealed that 10-15% of the 300 most abundant translation products are differentially synthesized from RNA obtained at various cell cycle stages. RNAs that direct the synthesis of alpha- and beta-tubulins and that hybridize to cloned alpha- and beta-tubulin probes accumulate coordinately during the predivision period of the cell cycle, reaching peak levels before or during division.

Isolation and genetic characterization of a mutation affecting ribosomal resistance to cycloheximide in Tetrahymena.

A dominant mutation at a new locus affecting resistance to cycloheximide has been isolated by exploiting a synergistic relationship with a previously known mutation for cycloheximide resistance in Tetrahymena. The new mutation (ChxB) was induced in a line homozygous for ChxA and was recovered from that background by a new technique termed interrupted genomic exclusion. Segregation data from the interrupted genomic exclusion suggest that ChxA and ChxB are separate, linked loci showing 30% recombination.

Cycloheximide resistance can be mediated through either ribosomal subunit.

Two cycloheximide-resistant mutants of Tetrahymena thermophila were analyzed to determine the site of their cycloheximide resistance. The mutations in both strains had been previously shown to be genetically dominant and located at separate loci (denoted Chx-A and Chx-B). Strains carrying these mutations were readily distinguished by the extent to which they were resistant to the drug.