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M Rosbash

Publications and source records attributed to M Rosbash.

At least 127 records · Page 7Linked to original sources
PubMed1990

Universally conserved and yeast-specific U1 snRNA sequences are important but not essential for U1 snRNP function.

To study the contribution of the large, 568-nucleotide yeast (Saccharomyces cerevisiae) U1 snRNA to pre-mRNA splicing, we generated mutations in two regions of the molecule and introduced each mutant gene back into yeast as the sole copy of the U1 snRNA gene. We mutagenized the "A loop," a subregion highly conserved in primary sequence in all U1 snRNA molecules analyzed to date. We also mutagenized a portion of the yeast core subdomain, a region conserved in primary and secondary structure among several yeast species but absent from the much smaller metazoan U1 molecule. Surprisingly, mutations in these two regions had little or no effect on growth rate, yet several of them affected an inefficiently spliced reporter gene construct. In addition, combinations of mutants in both regions gave rise to reduced growth rates. Using the latter assay, we confirmed some of the proposed secondary structure of the yeast core domain. The experiments indicate that both regions contribute to U1 snRNP activity but that mutations in a single region do not have a substantial effect on growth rate because U1 snRNP activity is not rate-limiting for growth.

Base Sequence↗
PubMed1990

The yeast PRP6 gene encodes a U4/U6 small nuclear ribonucleoprotein particle (snRNP) protein, and the PRP9 gene encodes a protein required for U2 snRNP binding.

PRP6 and PRP9 are two yeast genes involved in pre-mRNA splicing. Incubation at 37 degrees C of strains that carry temperature-sensitive mutations at these loci inhibits splicing, and in vivo experiments suggested that they might be involved in commitment complex formation (P. Legrain and M. Rosbash, Cell 57:573-583, 1989). To examine the specific role that the PRP6 and PRP9 products may play in splicing or pre-mRNA transport to the cytoplasm, we have characterized in vitro splicing and spliceosome assembly in extracts derived from prp6 and prp9 mutant strains. We have also characterized RNAs that are specifically immunoprecipitated with the PRP6 and PRP9 proteins. Both approaches indicate that PRP6 encodes a U4/U6 small nuclear ribonucleoprotein particle (snRNP) protein and that the PRP9 protein is required for a stable U2 snRNP-substrate interaction. The results are discussed with reference to the previously observed in vivo phenotypes of these mutants.

Genes, Fungal↗
PubMed1990

Requirement for period gene expression in the adult and not during development for locomotor activity rhythms of imaginal Drosophila melanogaster.

Mutations at the period (per) locus of Drosophila melanogaster disrupt the circadian rhythm of adult locomotor activity. Molecular studies have shown that this gene is expressed primarily at the embryonic, pupal and adult stages. We have used conditional per mutants to infer the stages of development during which per expression is required for adult rhythmicity. In experiments carried out with germline transformants in which the arrhythmic per01 allele has been transformed with a heat-shock protein 70 promoter-driven per gene (hsp-per transformants) we find that per expression in the adult is both necessary and sufficient for imaginal rhythms. Results obtained with existing per alleles and other per transformant strains that behave as conditional per mutants are consistent with those obtained with these molecularly engineered conditional mutants. Using hsp-per transformants we have found that the per gene product is apparently required only at the time of manifestation of rhythmicity, and can rescue the host's arrhythmic phenotype even when supplied many days after transfer to constant darkness. We present evidence suggesting that it is necessary for pacemaker function itself, rather than being involved in a process that couples the activity of the pacemaker to the output pathway. The levels of per transcript and the abundance and tissue distribution of its protein product observed in hsp-per transformants exposed to different temperature regimes are described. An initial report of some of these results has been published previously (Ewer et al., 1988).

Activity Cycles↗
PubMed1990

Circadian fluctuations of period protein immunoreactivity in the CNS and the visual system of Drosophila.

When the protein encoded by the period (per) gene, which influences circadian rhythms in Drosophila melanogaster, was labeled with an anti-per antibody in adult flies sectioned at different times of day, regular fluctuations in the intensity of immunoreactivity were observed in cells of the visual system and central brain. These fluctuations persisted in constant darkness. Time courses of the changing levels of staining were altered in the per-short mutant: in light/dark cycles, the phase was earlier than in wild-type, and in constant darkness the period was shorter. In a per-long mutant and in behaviorally subnormal germline transformants (involving transduced per DNA), staining intensities were much fainter than in wild-type. Factors involved in initiating or maintaining the per protein cycling were investigated by examining the immunoreactivity in visual system mutants and by exposing wild-type flies to altered light/dark regimes. These genetic and environmental manipulations affected the expression of the per protein in ways that usually parallelled their effects on circadian behaviors.

Animals↗
PubMed1989

Identification of functional U1 snRNA-pre-mRNA complexes committed to spliceosome assembly and splicing.

Although both U1 and U2 snRNPs have been implicated in the splicing process, their respective roles in the earliest stages of intron recognition and spliceosome assembly are uncertain. To address this issue, we developed a new strategy to prepare snRNP-depleted splicing extracts using Saccharomyces cerevisiae cells conditionally expressing U1 or U2 snRNP. Complementation analyses and chase experiments show that a stable complex, committed to the splicing pathway, forms in the absence of U2 snRNP. U1 snRNP and a substrate containing both a 5' splice site and a branchpoint sequence are required for optimal formation of this commitment complex. We developed new gel electrophoresis conditions to identify these committed complexes and to show that they contain U1 snRNA. Chase experiments demonstrated that these complexes are functional intermediates in spliceosome assembly and splicing. Our results have implications for the process of splice site selection.

Base Sequence↗
PubMed1989

Mutational analysis of the interactions between U1 small nuclear RNA and pre-mRNA of yeast.

In recent experiments we have used the power of yeast genetics to study U1 small nuclear RNA (snRNA): pre-messenger RNA (pre-mRNA) base pairing interactions [Séraphin et al. EMBO J. 7 (1988) 2533-2538]. Here we extend these observations to other potential U1 snRNA: pre-mRNA pairings. We show that several U1 snRNA mutants are viable. Using these U1 mutant strains we demonstrate further a base-pairing interaction between U1 snRNA position 3 and intron position 6. However, this interaction is only detected with a poor splicing substrate containing branchpoint mutations. These results provide information on the mechanism of 5' splice site-branch point interaction. We also propose several models which may explain why the sequence of the 5' end of the U1 snRNA is conserved among organisms as divergent as man and yeast.

Animals↗
PubMed1989

Some cis- and trans-acting mutants for splicing target pre-mRNA to the cytoplasm.

We designed a strategy to identify splicing factors that act by preventing pre-mRNA transport into the cytoplasm. A yeast synthetic intron was inserted into a lacZ gene so that only the pre-mRNA could be translated to produce beta-galactosidase activity. Deletion of either of the 5' splice junction sequence GUAUGU and the branchpoint sequence UACUAAC resulted in a dramatic increase in pre-mRNA translation, indicating its cytoplasmic localization. In rna6 and rna9 mutant strains assayed at the nonpermissive temperature, splicing inhibition occurred simultaneously with a large increase in pre-mRNA translation. Similarly, a point mutation in U1 snRNA decreased splicing efficiency and increased pre-mRNA translation. From these results, we conclude that early acting factors, probably including U1 snRNA, and the RNA6 and RNA9 gene products, interact in vivo with the 5' splice junction and the branchpoint sequence to commit the pre-mRNA to the splicing pathway, thereby preventing its transport to the cytoplasm.

Amino Acid Sequence↗
PubMed1989

Sequence requirements for branch formation in a group II self-splicing intron.

Evidence is presented for the existence of a specific intron-intron interaction, necessary for the formation of the branched product in the self-splicing reaction of a group II yeast mitochondrial intron. Trans-splicing reactions involving two RNA molecules (5' exon with covalently linked regions of intron and intron with covalently linked 3' exon) show that the presence of portions of intron domain I on the 5' molecule is necessary for the formation of branched products which are not seen with shorter 5' molecules. Modification/interference reactions show regions necesary for branch-formation and support a major role for specific regions of intron domain I. Further experiments, utilizing a truncated 3' molecule that is missing the conserved branchpoint nucleotide, indicate that domain VI may be required for a successful domain I interaction. A model for the formation of a proper branched structure includes implications for both cis and trans configurations.

Base Sequence↗
PubMed1989

An antibody to the Drosophila period protein recognizes circadian pacemaker neurons in Aplysia and Bulla.

The molecular mechanisms of the pacemakers underlying circadian rhythms are not well understood. One molecule that presumably functions in the circadian clock of Drosophila is the product of the period (per) gene, which dramatically affects biological rhythms when mutated. An antibody specific for the per protein labels putative circadian pacemaker neurons and fibers in eyes of two marine gastropods, Aplysia and Bulla. As was found for the Drosophila per protein, there is a daily rhythm in the levels of the per-like antigen in Aplysia eyes. Thus, certain molecular features of the per protein, as well as aspects of the temporal regulation of its expression, may be conserved in circadian pacemakers of widely divergent species.

Animals↗
PubMed1989

The disconnected visual system mutations in Drosophila melanogaster drastically disrupt circadian rhythms.

Mutations at the disconnected (disco) locus in Drosophila melanogaster cause cultures of this insect to eclose in an essentially arrhythmic manner and also nearly eliminate free-running circadian rhythms of locomotor activity. Yet disco mutants are not totally light-insensitive: Whereas they performed very poorly in tests of certain behavioral responses to visual stimuli, they were able to exhibit "forced" periodic locomotor activity under conditions of light-dark cycling. We discuss these results in the context of (1) the dispensability of this insect's external photoreceptors for entrainment of its circadian pacemaker, and (2) possible disco-induced abnormalities in the connections of extraocular photoreceptors to their targets in the central nervous system and/or abnormalities in the targets themselves--which presumably include elements of the fly's circadian clock.

Animals↗
PubMed1989

Expression of a Drosophila mRNA is under circadian clock control during pupation.

Rhythmic eclosion of Drosophila adults requires per gene function. We have found that a previously identified 0.9 kb RNA transcribed from DNA adjacent to per becomes abundantly expressed during pupation, just prior to eclosion. The daily synchronized emergence of young adults, coupled with a subsequent rapid decay of the transcript, is responsible for what previously appeared to be cycling of the 0.9 kb RNA in adults. In situ hybridization analyses localize the 0.9 kb transcript to the epidermis of newly eclosed adults. Conceptual translation of genomic DNA and cDNA sequences predicts that the 0.9 kb transcript produces a 261 amino acid protein containing a putative signal sequence for membrane transport at its amino terminus. Pupae that reach the same stage of development at slightly different times of day show a subsequent synchronized rise in 0.9 kb RNA levels, indicating that the expression of this transcript is under circadian clock control.

Amino Acid Sequence↗
PubMed1989

A new mutation at the period locus of Drosophila melanogaster with some novel effects on circadian rhythms.

A new period mutation has been induced and characterized in D. melanogaster. It causes flies to be apparently arrhythmic in tests of locomotor activity and thus is superficially similar to the original per01 mutant. Yet, the new "zero" allele, per04, has some novel properties and effects: Behaviorally, per04 adults often exhibit weak, long-period rhythms of locomotor activity in constant darkness; this low-frequency rhythmicity usually was not obvious in the analog behavioral records but was readily revealed by spectral analyses. These treatments of the data also extracted hidden high-frequency (ultradian) rhythms in many of the behavioral records, of the type associated with per01 and other per-nulls. The wide range of periodicities exhibited by different per04-expressing flies implies the expression of multiple oscillatory modes by this mutant. The new mutation also leads to a tendency for flies to be hyperactive during activity monitoring and is thus dissimilar to the other arrhythmic variants in the per gene but similar to the effects of a deletion of the locus. During light:dark cycling, per04 adults once more behave differently from other per0's and in fact tend to resemble wild-type flies in these conditions. The new mutation is not caused by the same nucleotide substitution that created a stop codon in the original arrhythmic per mutant and, as it turns out, per02 and per03 as well. per04 is also not a null variant at the transcriptional level; but it leads to an anomalous form of per mRNA, which is smaller than the normal 4.5 kb species encoded by this clock gene.

Alleles↗
PubMed1988

Interspecific comparison of the period gene of Drosophila reveals large blocks of non-conserved coding DNA.

We have cloned and sequenced the coding region of the period (per) gene from Drosophila pseudoobscura and D. virilis. A comparison with that of D. melanogaster reveals that the conceptual translation products consist of interspersed blocks of conserved and non-conserved amino acid sequence. The non-conserved portion, comprising approximately 33% of the protein sequence, includes the perfect Thr-Gly repeat of D. melanogaster, which is absent from the D. pseudoobscura and D. virilis proteins. Based on these observations and cross-species transformation experiments, we suggest that the interspecific variability in the per primary amino acid sequence contributes to the control of species-specific behaviors.

Amino Acid Sequence↗
PubMed1988

The period gene of Drosophila carries species-specific behavioral instructions.

We have analyzed and compared the circadian locomotor activity rhythms of Drosophila melanogaster and D.pseudoobscura. The rhythms of D.pseudoobscura are stronger and the periods shorter than those of D.melanogaster. We have also transformed D.melanogaster flies with a hybrid gene containing the coding region of the D.pseudoobscura period (per) gene. Behavioral assays of flies containing this hybrid gene show that the per protein encoded by the D.pseudoobscura per gene is able to rescue the rhythmic deficiencies of arrhythmic, pero1 D.melanogaster. More important, the rhythms of some of these strains are stronger and the periods shorter than those of D.melanogaster (and those of transformants which carry the equivalent D.melanogaster per gene construct) and hence resemble those of D.pseudoobscura. The results suggest that the primary amino acid sequence of the per gene encodes species-specific behavioral instructions that are detectable when only the per gene is transferred to a different species.

Animals↗
PubMed1988

An inducible promoter fused to the period gene in Drosophila conditionally rescues adult per-mutant arrhythmicity.

The period (per) gene of Drosophila melanogaster is involved in the expression of circadian rhythms of locomotor activity in adult flies. Molecular studies of per (reviewed in ref. 2) have shown that the transcribed and translated products of this gene are present primarily at the embryonic, pupal and adult stages. Here we describe experiments with arrhythmic per mutants bearing an inducible form of this gene which indicate that strongly rhythmic adult behaviour can be obtained only if per expression is induced in the adult, independent of its history of expression earlier in development. Thus per-mutant locomotor-activity phenotypes seem not to result from abnormalities in the development of neural structures or in physiological processes that may be required at pre-adult stages for the expression of this circadian rhythm. Moreover, the action of per after light:dark cycle entrainment seems to be sufficient for activity rhythms to be exhibited in constant darkness; this suggests further that the per product is required only during the time that the rhythmic behaviour is being manifested. Our strategy used a heat-shock gene promotor fused to per coding sequences to obtain conditional gene expression. Heat-shock promoter-driven genes have previously been used to study the mode of action and tissue specificity of a variety of Drosophila genes; our experiments on circadian rhythms demonstrate the use of such gene constructions for the temporal manipulation of genes whose phenotypes, behavioural and otherwise, affect whole organisms.

Animals↗
PubMed1988

A U1 snRNA:pre-mRNA base pairing interaction is required early in yeast spliceosome assembly but does not uniquely define the 5' cleavage site.

We analyzed the effects of suppressor mutations in the U1 snRNA (SNR19) gene from Saccharomyces cerevisiae on the splicing of mutant pre-mRNA substrates. The results indicate that pairing between U1 snRNA and the highly conserved position 5 (GTATGT) of the intron occurs early in spliceosome assembly in vitro. This pairing is important for efficient splicing both in vitro and in vivo. However, pairing at position 5 does not appear to influence 5' splice site selection in vivo, indicating that the previously described U1 snRNA:5' splice junction base pairing interaction is not sufficient to define the 5' cleavage site.

Introns↗
PubMed1988

Antibodies to the period gene product of Drosophila reveal diverse tissue distribution and rhythmic changes in the visual system.

Polyclonal antibodies were prepared against the period gene product, which influences biological rhythms in D. melanogaster, by using small synthetic peptides from the per sequence as immunogens. The peptide that elicited the best antibody reagent was a small domain near the site of the pers (short period) mutation. Specific immunohistochemical staining was detected in a variety of tissue types: the embryonic CNS; a few cell bodies in the central brain of pupae; these and other cells in the central brain of adults, as well as imaginal cells in the eyes, optic lobes, and the gut. The intensity of per-specific staining in the visual system was found to oscillate, defining a free-running circadian rhythm with a peak in the middle of the night.

Animals↗