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M Rosbash

Publications and source records attributed to M Rosbash.

At least 55 records · Page 3Linked to original sources
PubMed1998

A comprehensive biochemical and genetic analysis of the yeast U1 snRNP reveals five novel proteins.

The U1 snRNP is essential for recognition of the pre-mRNA 5'-splice site and the subsequent assembly of the spliceosome. Yeast U1 snRNP is considerably more complex than its metazoan counterpart, which suggests possible differences between yeast and metazoa in early splicing events. We have comprehensively analyzed the composition of yeast U1 snRNPs using a combination of biochemical, mass spectrometric, and genetic methods. We demonstrate the specific association of four novel U1 snRNP proteins, Snu71p, Snu65p, Nam8p, and Snu56p, that have no known metazoan homologues. A fifth protein, Npl3p, is an abundant cellular component that reproducibly co-purifies with the U1 snRNP, but its association is salt-sensitive. Therefore, we are unable to establish conclusively whether it binds specifically to the U1 snRNP. Interestingly, Nam8p and Npl3p were previously assigned functions in (pre-m)RNA-metabolism; however, so far, no association with U1 snRNP has been demonstrated or proposed. We also show that the yeast SmB protein is a U1 snRNP component. Yeast U1 snRNP therefore contains 16 different proteins, including seven snRNP core proteins, three homologues of the metazoan U1 snRNP-specific proteins, and six yeast-specific U1 snRNP proteins. We have simultaneously continued the characterization of additional mutants isolated in a synthetic lethal (MUD) screen for genes that functionally cooperate with U1 snRNA. Consistent with the biochemical results, mud10, mud15, and mud16 are alleles of SNU56, NAM8, and SNU65, respectively. mud10 and mud15 affect the in vivo splicing efficiency of noncanonical introns. Moreover, mud10p strongly affects the in vitro formation of splicing complexes, and extracts from the mud15 strain contain a U1 snRNP that migrates aberrantly on native gels. Finally, we show that Nam8p/Mud15p contributes to the stability of U1 snRNP.

Amino Acid Sequence↗
PubMed1997

Post-transcriptional regulation contributes to Drosophila clock gene mRNA cycling.

The period (per) and timeless (tim) genes are intimately involved in the generation and maintenance of Drosophila circadian rhythms. Both genes are expressed in a circadian manner, and the two proteins (PER and TIM) participate in feedback regulation which contributes to the mRNA oscillations. Previous studies indicate that the circadian regulation is in part transcriptional. To investigate quantitative features of per and tim transcription, we analyzed the in vivo transcription rate in fly-head nuclei with a nuclear run-on assay. The results show a robust transcriptional regulation, which is similar but not identical for the two genes. In addition, per mRNA levels are regulated at a post-transcriptional level. This regulatory mode makes a major contribution to the per mRNA oscillations from a previously described per transgenic strain as well as to the mRNA oscillations of a recently identified Drosophila circadianly regulated gene (Crg-1). The data show that circadian mRNA oscillations can take place without evident transcriptional regulation.

Animals↗
PubMed1997

The yeast nucleoporin rip1p contributes to multiple export pathways with no essential role for its FG-repeat region.

The FG-repeat domain of the yeast Rip1 protein (Rip1p) was identified initially as a possible target for the nuclear export signal (NES) of the HIV-1 Rev protein in a yeast two-hybrid assay. Rip1p is inessential, associated with nuclear pore complexes, and structurally related to the FG-nucleoporin family of pore proteins. It contributes to HIV-1 Rev-mediated RNA export and is also important for the export of heat shock RNAs at 42 degrees C. We show here that Rip1p is essential for the export of heat shock RNAs, and this function is fulfilled by the unique carboxyl terminus of Rip1p with no substantial contribution from the FG-repeat region. Genetic interactions between Rip1p and the RNA export mediator Gle1p are described, which support a role of the carboxyl terminus of Rip1p in poly(A)+ RNA export. Finally, this domain of Rip1p also contributes to Rev-mediated RNA export. The data suggest that Rip1p promotes the nuclear export of different classes of substrates by contributing to optimal pore function.

Amino Acid Sequence↗
PubMed1997

The importin-beta family member Crm1p bridges the interaction between Rev and the nuclear pore complex during nuclear export.

BACKGROUND: The human immunodeficiency virus (HIV-1) uses the viral protein Rev to regulate gene expression by promoting the export of unspliced and partially spliced viral transcripts. Rev has been shown to function in a variety of organisms, including Saccharomyces cerevisiae. The export activity of Rev depends on a nuclear export signal (NES), which is believed to interact either directly or indirectly with the nuclear pore complex to carry out its export function. Crm1p is a member of the importin-beta protein family, other members of which are known to be directly involved in nuclear import. Crm1p has recently been shown to contribute to nuclear export in vertebrate systems. Here, we have studied this mechanism of nuclear to cytoplasmic transport. RESULTS: Viable mis-sense mutations in the CRM1 gene substantially reduced or eliminated the biological activity of Rev in S. cerevisiae, providing strong evidence that Crm1p also contributes to transport of Rev NES-containing proteins and ribonucleoproteins in this organism. Crm1p interacted with FG-repeat-containing nuclear pore proteins as well as Rev, and we have demonstrated that the previously described two-hybrid interaction between Rev and the yeast nuclear pore protein Rip1p is dependent on wild-type Crm1p. CONCLUSIONS: We conclude that Crm1p interacts with the Rev NES and nuclear pore proteins during delivery of cargo to the nuclear pore complex. Our findings also agree well with current experiments on Crm1p orthologs in Schizosaccharomyces pombe and in vertebrate systems.

Biological Transport↗
PubMed1997

A new gene encoding a putative transcription factor regulated by the Drosophila circadian clock.

Circadian rhythms of locomotor activity and eclosion in Drosophila depend upon the reciprocal autoregulation of the period (per) and timeless (tim) genes. As part of this regulatory loop, per and tim mRNA levels oscillate in a circadian fashion. Other cycling transcripts may participate in this central pacemaker mechanism or represent outputs of the clock. In this paper, we report the isolation of Crg-1, a new circadianly regulated gene. Like per and tim transcript levels, Crg-1 transcript levels oscillate with a 24 h period in light:dark (LD) conditions, with a maximal abundance at the beginning of the night. These oscillations persist in complete darkness and depend upon per and tim proteins. The putative CRG-1 proteins show some sequence similarity with the DNA-binding domain of the HNF3/fork head family of transcription factors. In the adult head, in situ hybridization analysis reveals that per and Crg-1 have similar expression patterns in the eyes and optic lobes.

Alternative Splicing↗
PubMed1997

Identification and characterization of a yeast homolog of U1 snRNP-specific protein C.

U1C is one of the three human U1 small nuclear ribonucleoprotein (snRNP)-specific proteins and is important for efficient complex formation between U1 snRNP and the pre-mRNA 5' splice site. We identified a hypothetical open reading frame in Saccharomyces cerevisiae as the yeast homolog of the human U1C protein. The gene is essential, and its product, YU1C, is associated with U1 snRNP. YU1C depletion gives rise to normal levels of U1 snRNP and does not have any detectable effect on U1 snRNP assembly. YU1C depletion and YU1C ts mutants affect pre-mRNA splicing in vivo, and extracts from these strains form low levels of commitment complexes and spliceosomes in vitro. These experiments indicate a role for YU1C in snRNP function. Structure probing with RNases shows that only the U1 snRNA 5' arm is hypersensitive to RNase I digestion when YU1C is depleted. Similar results were obtained with YU1C ts mutants, indicating that U1C contributes to a proper 5' arm structure prior to its base pairing interaction with the pre-mRNA 5' splice site.

Amino Acid Sequence↗
PubMed1997

The splicing factor BBP interacts specifically with the pre-mRNA branchpoint sequence UACUAAC.

The yeast splicing factor BBP (branchpoint bridging protein) interacts directly with pre-mRNA at or very near the highly conserved branchpoint sequence UACUAAC within the commitment complex. We also show that the recombinant protein recognizes the UACUAAC sequence. Therefore, BBP is also an acronym for branchpoint binding protein. The mammalian splicing factor SF1 is a BBP ortholog (mBBP) and an E complex component, and also has branchpoint sequence specificity. The relative conservation of this region in yeast and mammals correlates well with the RNA-binding differences between BBP and mBBP, suggesting that BBP contributes to branchpoint sequence definition in both systems.

Binding Sites↗
PubMed1997

Cross-intron bridging interactions in the yeast commitment complex are conserved in mammals.

The commitment complex is the first defined step in the yeast (S. cerevisiae) splicing pathway. It contains U1 snRNP as well as Mud2p, which resembles human U2AF65. In a genetic screen, we identified the yeast gene MSL-5, which is a novel commitment complex component. Genetic and biochemical criteria indicate a direct interaction between Msl5p and both Mud2p and the U1 snRNP protein Prp40p. This defines a bridge between the two ends of the intron. Msl5p (renamed BBP for branchpoint bridging protein) has a mammalian ortholog, the splicing factor SF1. Our results show that SF1 interacts strongly with human U2AF65, and that SF1 is a bona fide E complex component. This implies that aspects of these novel cross-intron protein-protein interactions are conserved between yeast and mammals.

Animals↗
PubMed1997

A dynamic in vivo view of the HIV-I Rev-RRE interaction.

The export of pre-mRNAs coding for the structural genes of the human immunodeficiency virus type I depends on the interaction of the Rev protein with a highly structured viral RNA sequence, the Rev-responsive element (RRE). To gain information about the structure of the RRE and the determinants of the in vivo RRE-Rev interaction, we have analyzed the structure of the 351 nt RRE RNA within living yeast (Saccharomyces cerevisiae) by dimethyl sulfate probing with or without Rev. The in vivo structure in the absence of Rev is generally similar to the previously established solution structure. In addition, we observe a single hypermethylated guanine residue (G128), located within the Rev high-affinity binding site, in vitro as well as in vivo. The important homopurine interaction between residues 129 and 106 is required for the hyperreactivity, confirming its biological relevance. Expression of wild-type Rev leads to a protection of this region and to modifications of the RRE structure: the high-affinity site becomes further structured, and Stem IIA is destabilized. High-level expression of the oligomerization-defective mutant M4 protein leads to the same protections without destabilization of Stem IIA. Taken together with other observations, the data suggest that Rev captures the unusual conformation of the high-affinity site, followed by additional changes in the structure of the RRE.

Base Sequence↗
PubMed1997

A high affinity binding site for the HIV-1 nucleocapsid protein.

The nucleocapsid protein (NC) of HIV-1 is a small zinc finger protein that contributes to multiple steps of the viral life cycle, including the proper encapsidation of HIV RNA. This is accomplished through an interaction between NC and a region at the 5'-end of the RNA, defined as the Psi element. However, the specificity of NC for Psi or for RNA in general is not well understood. To study this problem, we used SELEX to identify high affinity RNA ligands that bind to NC. A 'winner' molecule (SelPsi), as well as a subregion of Psi RNA, were further characterized to understand the interaction between NC and SelPsi and its relationship to the interaction between NC and Psi. The comparison makes predictions about the sequence and structure of a high affinity binding site within the HIV-1 Psi element.

Base Sequence↗
PubMed1997

Temporal and spatial expression patterns of transgenes containing increasing amounts of the Drosophila clock gene period and a lacZ reporter: mapping elements of the PER protein involved in circadian cycling.

Rhythmic oscillations of the PER protein, the product of the Drosophila period (per) gene, in brain neurons of the adult fly are strongly involved in the control of circadian rhythms. We analyzed temporal and spatial expression patterns of three per-reporter fusion genes, which share the same 4 kb regulatory upstream region but contain increasing amounts of per's coding region fused in frame to the bacterial lacZ gene. The fusion proteins contained either the N-terminal half (SG), the N-terminal-two-thirds (BG), or nearly all (XLG) of the PER protein. All constructs led to reporter signals only in the known per-expressing cell types within the anterior CNS and PNS. Whereas the staining intensity of SG files was constantly high at different Zeitgeber times, the in situ signals in BG and XLG files cycled with approximately 24 hr periodicity in the PER-expressing brain cells in wild-type and per01 loss of function files. Despite the rhythmic fusion-gene expression within the relevant neurons of per01 BG files, their locomotor activity in light/dark cycling conditions and in constant darkness was identical to that of per01 controls, uncoupling protein cycling from rhythmic behavior. The XLG construct restored weak behavioral rhythmicity to (otherwise) per01 files, indicating that the C-terminal third of PER (missing in BG) is necessary to fulfill the biological function of this clock protein.

Animals↗
PubMed1997

Circadian cycling of a PERIOD-beta-galactosidase fusion protein in Drosophila: evidence for cyclical degradation.

The authors analyzed circadian features of two period-lacZ (per-lacZ) fusion genes in transgenic strains of Drosophila. Both genes manifest circadian fluctuations of mRNA levels, but fluctuations of only the larger chimeric protein are apparent. Fusion protein cycling is indistinguishable from the behavior of wild-type per protein (PER), including apparent temporal regulation of phosphorylation state. Several arguments indicate that the difference in the two constructs is proper regulation at the level of protein turnover: the smaller protein has much higher levels; a beta-galactosidase degradation products is visible in both strains but fails to manifest cycling, presumably due to a long half-life; and only the noncycling proteins accumulate as a function of adult age. The large cycling fusion protein also undergoes modest cycling in an arrhythmic per01 background. This is light dependent, resembles the regulation of the timeless protein (TIM) by light, and reflects a documented fusion protein-TIM interaction. The results are discussed with respect to the posttranscriptional regulation that is necessary for proper cycling of both PER and TIM as well as for clock function.

Aging↗
PubMed1996

Effect of constant light and circadian entrainment of perS flies: evidence for light-mediated delay of the negative feedback loop in Drosophila.

Light is the dominant environmental cue that provides temporal information to circadian pacemakers. In Drosophila melanogaster some period gene mutants have altered free-running circadian periods but entrain to 24 h light-dark cycles. To address the mechanism of light entrainment in Drosophila, we examined the effects of constant light on the period gene (per) and timeless gene (tim) products in wild-type and perS flies. The results indicate that light affects three features of the PER-TIM program: PER and TIM phosphorylation, PER and TIM accumulation, and per and tim RNA cycling. A post-transcriptional effect on the PER-TIM complex is the likely primary clock target, which then delays the subsequent decrease in per and tim RNA levels. This is consistent with a negative feedback loop, in which the PER-TIM complex contributes to the decrease in per and tim RNA levels, presumably at the transcriptional level. There are enhanced constant light effects on the perS mutant, which further support negative feedback as well as support its importance to entrainment of these flies to a 24 h cycle, far from their intrinsic period of 19 h. The perS mutant leads to a truncated protein accumulation phase and a subsequent premature perS RNA increase. A standard 24 h light-dark cycle delays the negative feedback circuit and extends the RNA and protein profiles, compensating for the accelerated RNA increase and restoring the rhythms to wild-type-like periodicity.

Animals↗
PubMed1996

Yeast pre-mRNA is composed of two populations with distinct kinetic properties.

As an approach to the study of yeast pre-mRNA splicing in vivo, we have examined properties of transcripts derived from a gal-UAS intron-containing fusion gene encoding RP51A and a series of its derivatives. RNA half-life measurements were carried out after transcription initiation was blocked by the addition of glucose. Pre-mRNA encoded by GalRP51A decayed with a half-life of approximately 6 min and was substantially polyadenylated, and transcripts derived from a nonspliced version of the same gene decayed with a similar half-life ( approximately 4 min). A comparison of the steady-state levels of these two transcripts suggests that the bulk of GalRP51A pre-mRNA is processed much more rapidly, with an average lifetime of about 2 s. We propose that this inferred population of rapidly processed molecules is spliced cotranscriptionally and that it is the principal precursor to GalRP51A mRNA. Although the pre-mRNA molecules detected are therefore unlikely to be the major splicing precursors, an in vivo assay suggests that they are likely to have bound splicing factors. They must then be spliced much more slowly than most primary transcripts, or not spliced at all and then degraded through a different cellular pathway. As a result of its comparatively long lifetime, this minor fraction of the pre-mRNA population makes up the majority of the steady-state level of GalRP51A pre-mRNA.

Base Sequence↗
PubMed1996

The yeast splicing factor Mud13p is a commitment complex component and corresponds to CBP20, the small subunit of the nuclear cap-binding complex.

The mechanism by which pre-mRNAs are initially recognized by the splicing machinery is not well understood. In the yeast system, commitment complexes are the earliest identified splicing complexes. They contain pre-mRNA, U1 snRNP, and the splicing factor Mud2p and probably correspond to the mammalian E complexes, which contain pre-mRNA, U1 snRNP, and the splicing factor U2AF. To identify other yeast commitment complex components, we have characterized mutant strains that are synthetic lethal with viable U1 snRNA mutations. We report here that MUD13 is a nonessential gene that encodes the yeast homolog of CBP20, the small subunit of the vertebrate nuclear cap-binding complex (CBC). Characterization of splicing in the delta-MUD13 strain and extract indicates that Mud13p is a yeast splicing factor and is the second identified non-snRNP commitment complex component. The observations also suggest that CBC interacts with other commitment complex components as well as with the substrate cap. Taken together with the accompanying results for a mammalian system, our data indicate that cap-binding proteins as well as the pre-mRNA cap contribute to early steps in spliceosome assembly.

Amino Acid Sequence↗
PubMed1996

A light-entrainment mechanism for the Drosophila circadian clock.

Biochemical studies indicate that the Drosophila timeless protein (Tim) is a stoichiometric partner of the period protein (Per) in fly head extracts. A Per-Tim heterodimeric complex explains the reciprocal autoregulation of the proteins on transcription. The complex is under clock control, and many circadian features of the Tim cycle resemble those of the Per cycle. However, Tim is rapidly degraded in the early morning or in response to light, releasing Per from the complex. The Per-Tim complex is a functional unit of the Drosophila circadian clock, and Tim degradation may be the initial response of the clock to light.

Animals↗
PubMed1996

The timSL mutant of the Drosophila rhythm gene timeless manifests allele-specific interactions with period gene mutants.

To identify new components of the Drosophila circadian clock, we screened chemically mutagenized flies for suppressors or enhancers of the long periods characteristic of the period (per) mutant allele perL. We isolated a novel mutant that maps to the rhythm gene timeless (tim). This novel allele, timSL, alters the temporal pattern of perL protein nuclear localization and restores temperature compensation to perL flies. timSL more generally manifests specific interactions with different per alleles. The identification of this first period-altering tim allele provides further evidence that TIM is a major component of the clock, and the allele-specific interactions with PER provide evidence that the PER/TIM heterodimer is a unit of circadian function. Although timSL fails to restore PER-L/TIM temperature insensitivity in yeast, it alters the TIM phosphorylation pattern during the late night. The effects on phosphorylation suggest that timSL functions as a partial bypass suppressor of perL and provide evidence that the TIM phosphorylation program contributes to the circadian timekeeping mechanism.

Alleles↗