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M Rosbash

Publications and source records attributed to M Rosbash.

At least 91 records · Page 5Linked to original sources
PubMed1994

A promoterless period gene mediates behavioral rhythmicity and cyclical per expression in a restricted subset of the Drosophila nervous system.

Transgenic flies carrying a 7.2 kb piece of DNA from the period (per) gene were analyzed for the presence of circadian locomotor activity rhythms and fluctuations of per-encoded mRNA and protein. The 5' end of this genomic fragment is within the first intron, which precedes the coding region. This promotorless fragment could rescue circadian behavioral rhythms and mediate spatial expression of PER in a subset of wild-type per cells within the CNS and PNS. In one behaviorally rhythmic line, PER protein was found in only "per lateral neurons." In the rhythmic transgenics, per mRNA and protein levels undergo circadian cycling, as previously described for wild type. Cycling of PER in brain cells of flies carrying the same 7.2 kb piece of per DNA under the control of a heat shock promoter corroborated the hypothesis that per's molecular cyclings and behavioral rhythmicity are causally related.

Animals↗
PubMed1994

mRNA nuclear export.

The export of mRNA from the nucleus to the cytoplasm is an essential step in the expression of genetic information in eukaryotes. It is an energy-dependent process and involves transport across the nuclear pores. It requires both cis-acting ribonucleoprotein particle signals and specific trans-acting factors. Although much remains to be learned, recent information has begun to define this pathway at both the cellular and biochemical levels and indicates that it is used as a key regulatory step by several viruses.

Animals↗
PubMed1993

Stabilization and ribosome association of unspliced pre-mRNAs in a yeast upf1- mutant.

Nonsense-mediated mRNA decay, the accelerated turnover of mRNAs transcribed from genes containing early nonsense mutations, is dependent on the product of the UPF1 gene in yeast. Mutations that inactivate UPF1 lead to the selective stabilization of mRNAs containing early nonsense mutations but have no effect on the half-lives of almost all other mRNAs. Since the transcripts of nonsense alleles are not typical cellular constituents, we sought to identify those RNAs that comprise normal substrates of the nonsense-mediated mRNA decay pathway. Many yeast pre-mRNAs contain early in-frame nonsense codons and we consider it possible that a role of this pathway is to accelerate the degradation of pre-mRNAs present in the cytoplasm. Consistent with this hypothesis, we find that, in a strain lacking UPF1 function, the CYH2, RP51B, and MER2 pre-mRNAs are stabilized 2- to 5-fold and are associated with ribosomes. We conclude that a major source of early nonsense codon-containing cytoplasmic transcripts in yeast is pre-mRNAs and that the UPF1 protein may be part of a cellular system that ensures that potentially deleterious nonsense fragments of polypeptides do not accumulate.

Gene Expression Regulation, Fungal↗
PubMed1993

PAS is a dimerization domain common to Drosophila period and several transcription factors.

Mutations in the period gene product (PER) can shorten or lengthen the circadian rhythms of Drosophila melanogaster, but its biochemical activity has not been established. PER contains a motif of approximately 270 amino acids whose function is unknown (termed PAS) and which is also present in three transcription factors of the basic-helix-loop-helix (bHLH) type, in the D. melanogaster single-minded gene product (SIM), and in both subunits of the mammalian dioxin receptor complex. We show here that the PER PAS functions in vitro as a novel protein dimerization motif and that it can mediate associations between different members of the PAS protein family. The dimerization efficiency is decreased by several missense mutations in the PAS domain, including the original perL mutation, which lengthens circadian periods from 24 h to 29 h (ref. 1). The results indicate that the PAS domain may function as a dimerization domain in both SIM and the dioxin receptor complex, and that PER may regulate circadian gene transcription partly by interacting with the PAS domain of bHLH--PAS-containing transcription factors.

Amino Acid Sequence↗
PubMed1993

Splice site choice and splicing efficiency are positively influenced by pre-mRNA intramolecular base pairing in yeast.

Many of the mechanisms that govern splice site selection and splice site partner assignment during pre-mRNA splicing are obscure. To address this problem, we analyzed the splicing of transcripts containing chimeric introns or splice site duplications derived from two natural yeast genes. Our experiments indicate that there are strong context effects that influence splicing efficiency and relative splice site strength. Cis-competition experiments showed that the context effects are not only local, as the source of the 3' splice site region influences 5' splice site selection and the source of the 5' splice site affects 3' splice site selection. A significant fraction of the long-range context effect appears to be due to base pairing between two intronic regions near the 5' splice site and branchpoint, an interaction that positively affects splicing efficiency as well as splice site selection.

Base Sequence↗
PubMed1993

Transfer of dye among salivary gland cells is not affected by genetic variations of the period clock gene in Drosophila melanogaster.

Larval salivary gland cells of Drosophila melanogaster were injected with a fluorescent dye to assess strengths of intercellular communication among such cells, as influenced by mutations at the period locus and by a per transgene. This clock gene had been reported to increase the extent of dye transfer when mutated such that it shortens the period of biological rhythms; the previous study also showed that a per-null mutant decreased the strength of transfer among salivary gland cells. Our re-examination of this feature of larval physiology--in observer-blind analyses, using the pers and per0 mutants as well as two per-normal strains--revealed no appreciable differences in extents of dye transfer among these four genotypes. These results are discussed in the context of emerging findings which suggest that the period gene's product controls pacemaker functioning as an intracellularly acting entity.

Animals↗
PubMed1993

An enhancer screen identifies a gene that encodes the yeast U1 snRNP A protein: implications for snRNP protein function in pre-mRNA splicing.

In an enhancer screen for yeast mutants that may interact with U1 small nuclear RNA (snRNA), we identified a gene that encodes the apparent yeast homolog of the well-studied human U1A protein. Both in vitro and in vivo, the absence of the protein has a dramatic effect on the activity of U1 snRNP containing the mutant U1 snRNA used in the screen. Surprisingly, the U1A gene is inessential in a wild-type U1 RNA background, as growth rate and the splicing of endogenous pre-mRNA transcripts are normal in these strains that lack the U1A protein. Even in vitro, the absence of the protein has little effect on splicing. On the basis of these observations, we suggest that a principal role of the U1A protein is to help fold or maintain U1 RNA in an active configuration.

Alleles↗
PubMed1993

Short artificial hairpins sequester splicing signals and inhibit yeast pre-mRNA splicing.

To examine the stability of yeast (Saccharomyces cerevisiae) pre-mRNA structures, we inserted a series of small sequence elements that generated potential RNA hairpins at the 5' splice site and branch point regions. We analyzed spliceosome assembly and splicing in vitro as well as splicing and nuclear pre-mRNA retention in vivo. Surprisingly, the inhibition of in vivo splicing approximately paralleled that of in vitro splicing. Even a 6-nucleotide hairpin could be shown to inhibit splicing, and a 15-nucleotide hairpin gave rise to almost complete inhibition. The in vitro results indicate that hairpins that sequester the 5' splice site have a major effect on the early steps of spliceosome assembly, including U1 small nuclear ribonucleoprotein binding. The in vivo experiments lead to comparable conclusions as the sequestering hairpins apparently result in the transport of pre-mRNA to the cytoplasm. The observations are compared with previous data from both yeast and mammalian systems and suggest an important effect of pre-mRNA structure on in vivo splicing.

Base Composition↗
PubMed1993

U1 small nuclear ribonucleoprotein particle-protein interactions are revealed in Saccharomyces cerevisiae by in vivo competition assays.

Two highly conserved regions of the 586-nucleotide yeast (Saccharomyces cerevisiae) U1 small nuclear RNA (snRNA) can be mutated or deleted with little or no effect on growth rate: the universally conserved loop II (corresponding to the metazoan A loop) and the yeast core region (X. Liao, L. Kretzner, B. Séraphin, and M. Rosbash, Genes Dev. 4:1766-1774, 1990). To examine the contribution of these regions to U1 small nuclear ribonucleoprotein particle (snRNP) activity, a competitor U1 gene, encoding a nonfunctional U1 snRNA molecule, was introduced into a number of strains carrying a U1 snRNA gene with loop II or yeast core mutations. The presence of the nonfunctional U1 gene lowered the growth rate of these mutant strains but not wild-type strains, consistent with the notion that mutant U1 RNAs are less active than wild-type U1 snRNAs. A detailed analysis of the U1 snRNA levels and half-lives in a number of merodiploid strains suggests that these mutant U1 snRNAs interact with U1 snRNP proteins less well than do their wild-type counterparts. Competition for protein factors during snRNP assembly could account for a number of previous observations in both yeast and mammalian cells.

Base Sequence↗
PubMed1992

Circadian oscillations in period gene mRNA levels are transcriptionally regulated.

The period (per) gene is involved in regulating circadian rhythms in Drosophila melanogaster. The per gene is expressed in a circadian manner, where fluctuations in per mRNA abundance are influenced by its own translation product, which also cycles in abundance. Since per gene expression is necessary for circadian rhythmicity, we sought to determine how certain features of this feedback loop operate. The results of this study reveal that fluctuations in per mRNA are primarily controlled by fluctuations in per gene transcription, that per mRNA has a relatively short half-life, and that sequences sufficient to drive per mRNA cycling are present in 1.3 kilobases of 5' flanking sequences. These and other results indicate that the per feedback loop has all of the basic properties necessary to be a component of a circadian oscillator.

Animals↗
PubMed1992

Requirements for U2 snRNP addition to yeast pre-mRNA.

The in vitro spliceosome assembly pathway is conserved between yeast and mammals as U1 and U2 snRNPs associate with the pre-mRNA prior to U5 and U4/U6 snRNPs. In yeast, U1 snRNP-pre-mRNA complexes are the first splicing complexes visualized on native gels, and association with U1 snRNP apparently commits pre-mRNA to the spliceosome assembly pathway. The current study addresses U2 snRNP addition to commitment complexes. We show that commitment complex formation is relatively slow and does not require ATP, whereas U2 snRNP adds to the U1 snRNP complexes in a reaction that is relatively fast and requires ATP or hydrolyzable ATP analogs. In vitro spliceosome assembly was assayed in extracts derived from strains containing several U1 sRNA mutations. The results were consistent with a critical role for U1 snRNP in early complex formation. A mutation that disrupts the base-pairing between the 5' end of U1 snRNA and the 5' splice site allows some U2 snRNP addition to bypass the ATP requirement, suggesting that ATP may be used to destabilize certain U1 snRNP:pre-mRNA interactions to allow subsequent U2 snRNP addition.

Adenosine Triphosphate↗
PubMed1992

Behavioral and molecular analyses suggest that circadian output is disrupted by disconnected mutants in D. melanogaster.

Mutations in the disconnected (disco) gene act to disrupt neural cell patterning in the Drosophila visual system. These mutations also affect adult locomotor activity rhythms, as disco flies are arrhythmic under conditions of constant darkness (DD). To determine the state of the circadian pacemaker in disco mutants, we constructed with pers double mutants (a short period allele of the period gene) and assayed their behavioral rhythms in light-dark cycles (LD), and their biochemical rhythms of period gene expression under both LD and DD conditions. The results demonstrate that disco flies are rhythmic, indicating that they have an active circadian pacemaker that can be entrained by light. They also suggest that disco mutants block or interfere with elements of the circadian system located between the central pacemaker and its outputs that mediate overt rhythms.

Activity Cycles↗
PubMed1992

A yeast splicing factor is localized in discrete subnuclear domains.

Digital imaging microscopy has been used to visualize the splicing protein PRP6p and three other yeast nuclear proteins. The results show that PRP6p is uniquely localized to discrete subnuclear regions. A combination of cytological and biochemical assays suggests that these sites can be saturated when the protein is overexpressed and likely correspond to the location of U4/U6 snRNPs. The observations indicate that some splicing components are located in discrete subregions of the yeast nucleus, similar to the situation described for the mammalian nucleus.

Blotting, Western↗
PubMed1992

Defects in mRNA 3'-end formation, transcription initiation, and mRNA transport associated with the yeast mutation prp20: possible coupling of mRNA processing and chromatin structure.

A temperature-sensitive lethal mutation in Saccharomyces cerevisiae, prp20-1, causes defects in several different steps in mRNA metabolism, including mRNA 3'-end formation, transcription initiation, and mRNA transport. Previous work has demonstrated that prp20 mutants are defective in actin pre-mRNA splicing. PRP20 is related, both in structure and function, to the RCC1 gene of mammals and the PIM1 gene of Schizosaccharomyces pombe, both of which appear to regulate entry into mitosis and chromosome condensation. In this report we demonstrate that, after a shift of prp20 mutants to the restrictive temperature, transcripts of several genes (CUP1, CYH2, and GAL10) are produced that extend 1-10 kb beyond their normal polyadenylation sites. The failure in 3'-end formation occurs within 1-2 min of the temperature shift. Transcription initiation also is disrupted, in that initiation sites upstream of the normal cap site are used. mRNA transport from nucleus to cytoplasm also is perturbed: In situ hybridization using an oligo(dT) probe demonstrates accumulation of poly(A) in the nucleus, consistent with the accumulation of longer bulk poly(A) (up to approximately 90-100 nucleotides) and with a failure to transport newly synthesized RNA to the cytoplasm. We demonstrate that prp20 and rna1 mutants are very similar, if not identical, with respect to each of these biochemical phenotypes. In light of the putative role of PRP20 in mitotic control, our results suggest a common step in that process and multiple steps in mRNA synthesis and maturation. We speculate that the perturbations in mRNA processing are the result of effects on the chromatin-nascent RNP-transcription complex or misregulation of a cell cycle component that modifies multiple mRNA-processing activities.

Base Sequence↗
PubMed1992

Drosophila homologs of two mammalian intracellular Ca(2+)-release channels: identification and expression patterns of the inositol 1,4,5-triphosphate and the ryanodine receptor genes.

We have identified and cloned portions of two Drosophila genes homologous to two classes of mammalian intracellular Ca(2+)-release channels, the ryanodine receptor and the inositol 1,4,5-triphosphate (IP3) receptor. The Drosophila ryanodine receptor gene (dry) encodes an approx. 15 kb mRNA. It is expressed in the mesoderm of early stage-9 embryos and subsequently in somatic muscles and their precursor cells. In adults, dry mRNA was detected in tubular muscles and at a lower level in neuronal tissues. Embryonic expression of the Drosophila IP3 receptor gene (dip) appears more dynamic and is associated with developing anterior sense organs. In adults, dip expression occurs in several tissues, and relatively high levels of dip mRNA in adult antennae suggest a role for this gene product during olfactory transduction.

Amino Acid Sequence↗
PubMed1992

Mapping the clock rhythm mutation to the period locus of Drosophila melanogaster by germline transformation.

The Clock (Clk) mutation shortens circadian rhythms of locomotor activity and eclosion from ca. 24 h to 22.5-23 h. Clk was previously mapped, by meiotic recombination, very close to the period(per) locus on the X chromosome. To determine whether Clk is a mutation within the per gene or if the former is separate from the latter, two overlapping genomic fragments were cloned from Clk flies to produce a per-containing 13.2 kb construct, per01 flies (which by themselves are arrhythmic)--when transformed with this construct--expressed short-period rhythms. This indicates that the Clk mutation is contained within this 13.2 kb region and is almost certainly a new "fast-clock" allele of per.

Alleles↗