RESUMEN
U6 small nuclear (sn)RNA is the shortest and most conserved snRNA in the spliceosome and forms a substantial portion of its active site. Unlike the other four spliceosomal snRNAs, which are synthesized by RNA polymerase (RNAP) II, U6 is made by RNAP III. To determine if some aspect of U6 function is incompatible with synthesis by RNAP II, we created a U6 snRNA gene with RNAP II promoter and terminator sequences. This "U6-II" gene is functional as the sole source of U6 snRNA in yeast, but its transcript is much less stable than U6 snRNA made by RNAP III. Addition of the U4 snRNA Sm protein binding site to U6-II increased its stability and led to formation of U6-IIâ¢Sm complexes. We conclude that synthesis of U6 snRNA by RNAP III is not required for its function and that U6 snRNPs containing the Sm complex can form in vivo. The ability to synthesize U6 snRNA with RNAP II relaxes sequence restraints imposed by intragenic RNAP III promoter and terminator elements and allows facile control of U6 levels via regulators of RNAP II transcription.
Asunto(s)
ARN Polimerasa II , Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , ARN Polimerasa II/genética , ARN Polimerasa II/metabolismo , Empalme del ARN , Secuencia de Bases , ARN Nuclear Pequeño/metabolismo , ARN Polimerasa III/genéticaRESUMEN
Human pre-mRNA splicing is primarily catalyzed by the major spliceosome, comprising five small nuclear ribonucleoprotein complexes, U1, U2, U4, U5, and U6 snRNPs, each of which contains the corresponding U-rich snRNA. These snRNAs are encoded by large gene families exhibiting significant sequence variation, but it remains unknown if most human snRNA genes are untranscribed pseudogenes or produce variant snRNAs with the potential to differentially influence splicing. Since gene duplication and variation are powerful mechanisms of evolutionary adaptation, we sought to address this knowledge gap by systematically profiling human U1, U2, U4, and U5 snRNA variant gene transcripts. We identified 55 transcripts that are detectably expressed in human cells, 38 of which incorporate into snRNPs and spliceosomes in 293T cells. All U1 snRNA variants are more than 1000-fold less abundant in spliceosomes than the canonical U1, whereas at least 1% of spliceosomes contain a variant of U2 or U4. In contrast, eight U5 snRNA sequence variants occupy spliceosomes at levels of 1% to 46%. Furthermore, snRNA variants display distinct expression patterns across five human cell lines and adult and fetal tissues. Different RNA degradation rates contribute to the diverse steady state levels of snRNA variants. Our findings suggest that variant spliceosomes containing noncanonical snRNAs may contribute to different tissue- and cell-type-specific alternative splicing patterns.
Asunto(s)
Empalme del ARN , ARN Mensajero/genética , ARN Nuclear Pequeño/genética , Empalmosomas/genética , Adulto , Emparejamiento Base , Secuencia de Bases , Fraccionamiento Celular/métodos , Exones , Feto , Células HEK293 , Humanos , Intrones , Anotación de Secuencia Molecular , Conformación de Ácido Nucleico , Especificidad de Órganos , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , ARN Mensajero/química , ARN Mensajero/metabolismo , ARN Nuclear Pequeño/química , ARN Nuclear Pequeño/metabolismo , Empalmosomas/química , Empalmosomas/metabolismoRESUMEN
Eukaryotes possess eight highly conserved Lsm (like Sm) proteins that assemble into circular, heteroheptameric complexes, bind RNA, and direct a diverse range of biological processes. Among the many essential functions of Lsm proteins, the cytoplasmic Lsm1-7 complex initiates mRNA decay, while the nuclear Lsm2-8 complex acts as a chaperone for U6 spliceosomal RNA. It has been unclear how these complexes perform their distinct functions while differing by only one out of seven subunits. Here, we elucidate the molecular basis for Lsm-RNA recognition and present four high-resolution structures of Lsm complexes bound to RNAs. The structures of Lsm2-8 bound to RNA identify the unique 2',3' cyclic phosphate end of U6 as a prime determinant of specificity. In contrast, the Lsm1-7 complex strongly discriminates against cyclic phosphates and tightly binds to oligouridylate tracts with terminal purines. Lsm5 uniquely recognizes purine bases, explaining its divergent sequence relative to other Lsm subunits. Lsm1-7 loads onto RNA from the 3' end and removal of the Lsm1 carboxy-terminal region allows Lsm1-7 to scan along RNA, suggesting a gated mechanism for accessing internal binding sites. These data reveal the molecular basis for RNA binding by Lsm proteins, a fundamental step in the formation of molecular assemblies that are central to eukaryotic mRNA metabolism.
Asunto(s)
Estabilidad del ARN/genética , Proteínas de Unión al ARN/genética , Proteínas de Saccharomyces cerevisiae/genética , Sitios de Unión/genética , Unión Proteica/genética , ARN/genética , Proteínas de Unión a Caperuzas de ARN/genética , Empalme del ARN/genética , ARN Mensajero/genética , ARN Nuclear Pequeño/genética , Ribonucleoproteínas Nucleares Pequeñas/genética , Saccharomyces cerevisiae/genética , Empalmosomas/genéticaRESUMEN
Removal of introns from precursor messenger RNA (pre-mRNA) and some noncoding transcripts is an essential step in eukaryotic gene expression. In the nucleus, this process of RNA splicing is carried out by the spliceosome, a multi-megaDalton macromolecular machine whose core components are conserved from yeast to humans. In addition to many proteins, the spliceosome contains five uridine-rich small nuclear RNAs (snRNAs) that undergo an elaborate series of conformational changes to correctly recognize the splice sites and catalyze intron removal. Decades of biochemical and genetic data, along with recent cryo-EM structures, unequivocally demonstrate that U6 snRNA forms much of the catalytic core of the spliceosome and is highly dynamic, interacting with three snRNAs, the pre-mRNA substrate, and >25 protein partners throughout the splicing cycle. This review summarizes the current state of knowledge on how U6 snRNA is synthesized, modified, incorporated into snRNPs and spliceosomes, recycled, and degraded.
Asunto(s)
Sitios de Empalme de ARN/genética , Empalme del ARN/genética , ARN Mensajero/genética , ARN Nuclear Pequeño/genética , Empalmosomas/metabolismo , Humanos , Conformación de Ácido Nucleico , ARN Polimerasa III/metabolismo , Procesamiento Postranscripcional del ARN/genética , ARN Nuclear Pequeño/biosíntesis , Saccharomyces cerevisiae/genéticaRESUMEN
Transcriptome studies on eukaryotic cells have revealed an unexpected abundance and diversity of noncoding RNAs synthesized by RNA polymerase II (Pol II), some of which influence the expression of protein-coding genes. Yet, much less is known about biogenesis of Pol II non-coding RNA than mRNAs. In the budding yeast Saccharomyces cerevisiae, initiation of non-coding transcripts by Pol II appears to be similar to that of mRNAs, but a distinct pathway is utilized for termination of most non-coding RNAs: the Sen1-dependent or "NNS" pathway. Here, we examine the effect on the S. cerevisiae transcriptome of conditional mutations in the genes encoding six different essential proteins that influence Sen1-dependent termination: Sen1, Nrd1, Nab3, Ssu72, Rpb11, and Hrp1. We observe surprisingly diverse effects on transcript abundance for the different proteins that cannot be explained simply by differing severity of the mutations. Rather, we infer from our results that termination of Pol II transcription of non-coding RNA genes is subject to complex combinatorial control that likely involves proteins beyond those studied here. Furthermore, we identify new targets and functions of Sen1-dependent termination, including a role in repression of meiotic genes in vegetative cells. In combination with other recent whole-genome studies on termination of non-coding RNAs, our results provide promising directions for further investigation.
Asunto(s)
ADN Helicasas/genética , ARN Helicasas/genética , ARN Polimerasa II/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Terminación de la Transcripción Genética , Regulación Fúngica de la Expresión Génica , Genoma Fúngico , Meiosis/genética , Proteínas Mutantes/genética , Proteínas Nucleares/genética , Fosfoproteínas Fosfatasas/genética , ARN no Traducido/genética , Proteínas de Unión al ARN/genética , Transducción de Señal , Transcriptoma/genética , Factores de Escisión y Poliadenilación de ARNm/genéticaRESUMEN
In this issue of Molecular Cell,Skourti-Stathaki et al. (2011) report that human Senataxin, like its yeast homolog Sen1, promotes termination by RNA polymerase II and resolves RNA/DNA duplexes formed during transcription. Their results may help uncover a cause of motor neuron degeneration.
RESUMEN
Base-pairing of U4 and U6 snRNAs during di-snRNP assembly requires large-scale remodeling of RNA structure that is chaperoned by the U6 snRNP protein Prp24. We investigated the mechanism of U4/U6 annealing in vitro using an assay that enables visualization of ribonucleoprotein complexes and faithfully recapitulates known in vivo determinants for the process. We find that annealing, but not U6 RNA binding, is highly dependent on the electropositive character of a 20 Å-wide groove on the surface of Prp24. During annealing, we observe the formation of a stable ternary complex between U4 and U6 RNAs and Prp24, indicating that displacement of Prp24 in vivo requires additional factors. Mutations that stabilize the U6 'telestem' helix increase annealing rates by up to 15-fold, suggesting that telestem formation is rate-limiting for U4/U6 pairing. The Lsm2-8 complex, which binds adjacent to the telestem at the 3' end of U6, provides a comparable rate enhancement. Collectively, these data identify domains of the U6 snRNP that are critical for one of the first steps in assembly of the megaDalton U4/U6.U5 tri-snRNP complex, and lead to a dynamic model for U4/U6 pairing that involves a striking degree of evolved cooperativity between protein and RNA.
Asunto(s)
ARN Nuclear Pequeño/metabolismo , Ribonucleoproteína Nuclear Pequeña U4-U6/metabolismo , Ribonucleoproteínas Nucleares Pequeñas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Secuencia de Bases , Unión Competitiva , Cinética , Modelos Moleculares , Datos de Secuencia Molecular , Mutación , Conformación de Ácido Nucleico , Unión Proteica , Estructura Terciaria de Proteína , ARN de Hongos/química , ARN de Hongos/genética , ARN de Hongos/metabolismo , ARN Nuclear Pequeño/química , ARN Nuclear Pequeño/genética , Ribonucleoproteína Nuclear Pequeña U4-U6/genética , Ribonucleoproteínas Nucleares Pequeñas/química , Ribonucleoproteínas Nucleares Pequeñas/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
The small nuclear RNA (snRNA) components of the spliceosome undergo many conformational rearrangements during its assembly, catalytic activation and disassembly. The U4 and U6 snRNAs are incorporated into the spliceosome as a base-paired complex within the U4/U6.U5 small nuclear ribonucleoprotein (tri-snRNP). U4 and U6 are then unwound in order for U6 to pair with U2 to form the spliceosome's active site. After splicing, U2/U6 is unwound and U6 annealed to U4 to reassemble the tri-snRNP. U6 rearrangements are crucial for spliceosome formation but are poorly understood. We have used single-molecule Förster resonance energy transfer and unwinding assays to identify interactions that promote U4/U6 unwinding and have studied their impact in yeast. We find that U4/U6 is efficiently unwound using DNA oligonucleotides by coupling unwinding of U4/U6 stem II with strand invasion of stem I. Unwinding is stimulated by the U6 telestem, which transiently forms in the intact U4/U6 RNA complex. Stabilization of the telestem in vivo results in accumulation of U4/U6 di-snRNP and impairs yeast growth. Our data reveal conserved mechanisms for U4/U6 unwinding and indicate telestem dynamics are critical for tri-snRNP assembly and stability.
Asunto(s)
ARN de Hongos/química , ARN Nuclear Pequeño/química , Saccharomyces cerevisiae/fisiología , Emparejamiento Base , Cinética , Estabilidad del ARN , ARN Bicatenario/químicaRESUMEN
The cycle of spliceosome assembly, intron excision, and spliceosome disassembly involves large-scale structural rearrangements of U6 snRNA that are functionally important. U6 enters the splicing pathway bound to the Prp24 protein, which chaperones annealing of U6 to U4 RNA to form a U4/U6 di-snRNP. During catalytic activation of the assembled spliceosome, U4 snRNP is released and U6 is paired to U2 snRNA. Here we show that point mutations in U4 and U6 that decrease U4/U6 base-pairing in vivo are lethal in combination. However, this synthetic phenotype is rescued by a mutation in U6 that alters a U6-Prp24 contact and stabilizes U2/U6. Remarkably, the resulting viable triple mutant strain lacks detectable U4/U6 base-pairing and U4/U6 di-snRNP. Instead, this strain accumulates free U4 snRNP, protein-free U6 RNA, and a novel complex containing U2/U6 di-snRNP. Further mutational analysis indicates that disruption of the U6-Prp24 interaction rather than stabilization of U2/U6 renders stable U4/U6 di-snRNP assembly nonessential. We propose that an essential function of U4/U6 pairing is to displace Prp24 from U6 RNA, and thus a destabilized U6-Prp24 complex renders stable U4/U6 pairing nonessential.
Asunto(s)
ARN Nuclear Pequeño/metabolismo , Ribonucleoproteína Nuclear Pequeña U4-U6/metabolismo , Ribonucleoproteínas Nucleares Pequeñas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Empalmosomas/metabolismo , Emparejamiento Base , Secuencia de Bases , Epistasis Genética , Complejos Multiproteicos/genética , Complejos Multiproteicos/metabolismo , Mutación , Conformación de Ácido Nucleico , Multimerización de Proteína , Empalme del ARN/genética , ARN de Hongos/química , ARN de Hongos/genética , ARN de Hongos/metabolismo , ARN Nuclear Pequeño/química , ARN Nuclear Pequeño/genética , Ribonucleoproteína Nuclear Pequeña U4-U6/química , Ribonucleoproteína Nuclear Pequeña U4-U6/genética , Saccharomyces cerevisiae/genéticaRESUMEN
In the yeast Saccharomyces cerevisiae, the essential nuclear helicase Sen1 is required for efficient termination of transcription of short noncoding RNA genes by RNA polymerase II. However, the mechanism by which Sen1 promotes transcription termination is not known. Prior biochemical studies on the Sen1 homolog from Schizosaccharomyces pombe showed that it can bind and unwind both DNA and RNA, but the S. pombe protein is not essential and has not been demonstrated to function in transcription. Furthermore, Sen1 from either yeast has not previously been expressed as a recombinant protein, due to its large molecular mass (252 kDa in S. cerevisiae). Here, we report the purification and characterization of the 89-kDa S. cerevisiae Sen1 helicase domain (Sen1-HD) produced in Escherichia coli. Sen1-HD binds single-stranded RNA and DNA with similar affinity in the absence of ATP, but it binds RNA more stably than DNA in the presence of ATP, apparently due to a slower translocation rate on RNA. Translocation occurs in the 5' to 3' direction, as for the S. pombe protein. When purified from E. coli at a moderate salt concentration, Sen1-HD was associated with short RNAs that are enriched for the trinucleotide repeat (CAN)4. We propose that Sen1 binds to RNAs and prevents their stable pairing with DNA, consistent with in vivo studies by others showing increased R-loop (RNA/DNA hybrid) formation when Sen1 activity is impaired by mutations. Our results are consistent with a model in which Sen1 promotes transcription termination by resolving R-loops.
Asunto(s)
ADN Helicasas/química , ADN de Hongos/química , ARN Helicasas/química , ARN de Hongos/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/enzimología , ADN Helicasas/genética , ADN Helicasas/metabolismo , ADN de Hongos/genética , ADN de Hongos/metabolismo , Estructura Terciaria de Proteína , ARN Helicasas/genética , ARN Helicasas/metabolismo , ARN de Hongos/genética , ARN de Hongos/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Especificidad por Sustrato/fisiologíaRESUMEN
The stepwise assembly of the highly dynamic spliceosome is guided by RNA-dependent ATPases of the DEAD-box family, whose regulation is poorly understood. In the canonical assembly model, the U4/U6.U5 triple snRNP binds only after joining of the U1 and, subsequently, U2 snRNPs to the intron-containing pre-mRNA. Catalytic activation requires the exchange of U6 for U1 snRNA at the 5' splice site, which is promoted by the DEAD-box protein Prp28. Because Prp8, an integral U5 snRNP protein, is thought to be a central regulator of DEAD-box proteins, we conducted a targeted search in Prp8 for cold-insensitive suppressors of a cold-sensitive Prp28 mutant, prp28-1. We identified a cluster of suppressor mutations in an N-terminal bromodomain-like sequence of Prp8. To identify the precise defect in prp28-1 strains that is suppressed by the Prp8 alleles, we analyzed spliceosome assembly in vivo and in vitro. Surprisingly, in the prp28-1 strain, we observed a block not only to spliceosome activation but also to one of the earliest steps of assembly, formation of the ATP-independent commitment complex 2 (CC2). The Prp8 suppressor partially corrected both the early assembly and later activation defects of prp28-1, supporting a role for this U5 snRNP protein in both the ATP-independent and ATP-dependent functions of Prp28. We conclude that the U5 snRNP has a role in the earliest events of assembly, prior to its stable incorporation into the spliceosome.
Asunto(s)
Empalme Alternativo/fisiología , ARN Helicasas DEAD-box/metabolismo , Ribonucleoproteína Nuclear Pequeña U4-U6/metabolismo , Ribonucleoproteína Nuclear Pequeña U5/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Adenosina Trifosfato/farmacología , Secuencia de Aminoácidos , Sustitución de Aminoácidos/fisiología , ARN Helicasas DEAD-box/genética , Modelos Moleculares , Datos de Secuencia Molecular , Multimerización de Proteína/efectos de los fármacos , Multimerización de Proteína/fisiología , ARN Nuclear Pequeño/fisiología , Ribonucleoproteína Nuclear Pequeña U4-U6/genética , Ribonucleoproteína Nuclear Pequeña U5/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Homología de Secuencia de Aminoácido , Empalmosomas/efectos de los fármacos , Empalmosomas/fisiologíaRESUMEN
Guanine nucleotide negatively regulates yeast inosine monophosphate dehydrogenase (IMPDH) mRNA synthesis by an unknown mechanism. IMPDH catalyzes the first dedicated step of GTP biosynthesis, and feedback control of its expression maintains the proper balance of purine nucleotides. Here we show that RNA polymerase II (Pol II) responds to GTP concentration. When GTP is sufficient, Pol II initiates transcription of the IMPDH gene (IMD2) at TATA box-proximal "G" sites, producing attenuated transcripts. When GTP is deficient, Pol II initiates at an "A" further downstream, circumventing the regulatory terminator to produce IMPDH mRNA. A major determinant for GTP concentration-dependent initiation at the upstream sites is the presence of guanine at the first and second positions of the transcript. Mutations in the Rpb1 subunit of Pol II and in TFIIB disrupt IMD2 regulation by altering start site selection. Thus, Pol II initiation can be regulated by the concentration of initiating nucleotide.
Asunto(s)
Regulación Fúngica de la Expresión Génica/efectos de los fármacos , Guanosina Trifosfato/farmacología , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/genética , Sitio de Iniciación de la Transcripción , Transcripción Genética/efectos de los fármacos , Regiones no Traducidas 5'/genética , Secuencia de Bases , Codón Iniciador/genética , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Genes Fúngicos , IMP Deshidrogenasa/genética , Modelos Genéticos , Datos de Secuencia Molecular , Mutación/genética , Nucleotidiltransferasas/genética , ARN Polimerasa II/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Secuencias Reguladoras de Ácidos Nucleicos/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
The purine nucleotides ATP and GTP are made from the common precursor inosine monophosphate (IMP). Maintaining the correct balance of these nucleotides for optimal cell growth is controlled in part by the enzyme IMP dehydrogenase (IMPDH), which catalyzes the first dedicated step of GTP biosynthesis. The regulation of IMPDH mRNA and protein levels in the yeast S. cerevisiae grown in liquid culture has been studied in some detail, but regulation of IMPDH protein under conditions of cellular crowding on a solid substrate has not been examined. Here, we report real-time, live-cell analysis of the accumulation of the Imd2 isoform of IMPDH in yeast cells forming a monolayer colony in a microfluidic device over a 50-hour time course. We observe two distinct phases of increased Imd2 accumulation: a guanine-insensitive phase early in outgrowth and a guanine-sensitive phase later, when cells become crowded. We show that the IMPDH inhibitor mycophenolic acid enhances both phases of increase. Deletion of a transcription attenuator upstream of the mRNA start site that decreases Imd2 mRNA synthesis in the presence of high GTP increases the baseline level of Imd2 protein 10-fold and abolishes guanine-sensitive but not guanine-insensitive induction. Our results suggest that at least two mechanisms of yeast Imd2 regulation exist, the known GTP-dependent attenuation of RNA polymerase II elongation and a GTP concentration-independent pathway that may be controlled by cell growth state. Live-cell analysis of IMPDH protein levels in a growing yeast colony confirms a known mechanism of regulation and provides evidence for an additional mode of regulation. IMPORTANCE: This study used live-cell microscopy to track changes in the level of a key enzyme in GTP nucleotide biosynthesis, inosine monophosphate dehydrogenase (IMPDH), during growth of a brewers yeast colony over 2 days in a microfluidic device. The results show that feedback regulation via transcription attenuation allows cells to adapt to nutrient limitation in the crowded environs of a yeast colony. They also identify a novel mode of regulation of IMPDH level that is not driven by guanine nucleotide availability.
Asunto(s)
Regulación Fúngica de la Expresión Génica , Guanosina Trifosfato , IMP Deshidrogenasa , Saccharomyces cerevisiae , IMP Deshidrogenasa/metabolismo , IMP Deshidrogenasa/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/crecimiento & desarrollo , Guanosina Trifosfato/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Ácido Micofenólico/farmacologíaRESUMEN
The essential splicing factor Prp24 contains four RNA Recognition Motif (RRM) domains, and functions to anneal U6 and U4 RNAs during spliceosome assembly. Here, we report the structure and characterization of the C-terminal RRM4. This domain adopts a novel non-canonical RRM fold with two additional flanking α-helices that occlude its ß-sheet face, forming an occluded RRM (oRRM) domain. The flanking helices form a large electropositive surface. oRRM4 binds to and unwinds the U6 internal stem loop (U6 ISL), a stable helix that must be unwound during U4/U6 assembly. NMR data indicate that the process starts with the terminal base pairs of the helix and proceeds toward the loop. We propose a mechanistic and structural model of Prp24's annealing activity in which oRRM4 functions to destabilize the U6 ISL during U4/U6 assembly.
Asunto(s)
ARN Nuclear Pequeño/metabolismo , Ribonucleoproteínas Nucleares Pequeñas/química , Proteínas de Saccharomyces cerevisiae/química , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Sitios de Unión , Secuencia Conservada , Modelos Moleculares , Ribonucleoproteínas Nucleares Pequeñas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
Proteins that bind the nascent transcript exiting RNA polymerase II can regulate transcription elongation. The essential Saccharomyces cerevisiae hnRNP protein Hrp1 is one such protein and participates in both cleavage and polyadenylation-coupled and Nrd1-Nab3-Sen1-dependent RNA polymerase II termination. Prior evidence that Hrp1 is a positive RNA polymerase II elongation factor suggests that its release from the elongation complex promotes termination. Here we report the effects of deletions and substitutions in Hrp1 on its autoregulation via an Nrd1-Nab3-Sen1-dependent transcription attenuator in the 5'-UTR of its mRNA and on the function of an Hrp1-dependent Nrd1-Nab3-Sen1 terminator in the SNR82 snoRNA gene. Deletion of either of two central RNA recognition motifs or either of the flanking low-sequence complexity domains is lethal. Smaller, viable deletions in the amino-terminal low-sequence complexity domain cause readthrough of both the HRP1 attenuator and SNR82 terminator. Substitutions that cause readthrough localized mostly to the RNA recognition motifs, although not always to the RNA-binding face. We found that autoregulation of Hrp1 mRNA synthesis is surprisingly robust, overcoming the expected lethal effects of the start codon and frameshift mutations via overexpression of the mRNA up to 40-fold. Our results suggest a model in which binding of attenuator or terminator elements in the nascent transcript by RNA recognition motifs 1 and 2 disrupts interactions between RNA recognition motif 2 and the RNA polymerase II elongation complex, increasing its susceptibility to termination.
Asunto(s)
Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Ribonucleoproteínas Nucleares Heterogéneas/metabolismo , Homeostasis , Proteínas Nucleares/genética , ARN Polimerasa II/genética , ARN Polimerasa II/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN no Traducido/genética , Proteínas de Unión al ARN/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
U6 RNA plays a critical role in pre-mRNA splicing. Assembly of U6 into the spliceosome requires a significant structural rearrangement and base-pairing with U4 RNA. In the yeast Saccharomyces cerevisiae, this process requires the essential splicing factor Prp24. We present the characterization and structure of a complex containing one of Prp24's four RNA recognition motif (RRM) domains, RRM2, and a fragment of U6 RNA. NMR methods were used to identify the preferred U6 binding sequence of RRM2 (5'-GAGA-3'), measure the affinity of the interaction, and solve the structure of RRM2 bound to the hexaribonucleotide AGAGAU. Interdomain contacts observed between RRM2 and RRM3 in a crystal structure of the free protein are not detectable in solution. A structural model of RRM1 and RRM2 bound to a longer segment of U6 RNA is presented, and a partial mechanism for Prp24's annealing activity is proposed.
Asunto(s)
ARN Nuclear Pequeño/química , ARN Nuclear Pequeño/metabolismo , Ribonucleoproteínas Nucleares Pequeñas/química , Proteínas de Saccharomyces cerevisiae/química , Secuencia de Bases , Sitios de Unión , Secuencia Conservada , ADN Helicasas/química , ADN Helicasas/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Estructura Terciaria de Proteína , ARN de Hongos/química , ARN de Hongos/metabolismo , Ribonucleoproteínas Nucleares Pequeñas/metabolismo , Ribonucleósido Difosfato Reductasa/química , Ribonucleósido Difosfato Reductasa/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
The highly conserved internal stem-loop (ISL) of U6 spliceosomal RNA is unwound for U4/U6 complex formation during spliceosome assembly and reformed upon U4 release during spliceosome activation. The U6 ISL is structurally similar to Domain 5 of group II self-splicing introns, and contains a dynamic bulge that coordinates a Mg++ ion essential for the first catalytic step of splicing. We have analyzed the causes of growth defects resulting from mutations in the Saccharomyces cerevisiae U6 ISL-bulged nucleotide U80 and the adjacent C67-A79 base pair. Intragenic suppressors and enhancers of the cold-sensitive A79G mutation, which replaces the C-A pair with a C-G pair, suggest that it stabilizes the ISL, inhibits U4/U6 assembly, and may also disrupt spliceosome activation. The lethality of mutations C67A and C67G results from disruption of base-pairing potential between U4 and U6, as these mutations are fully suppressed by compensatory mutations in U4 RNA. Strikingly, suppressor analysis shows that the lethality of the U80G mutation is due not only to formation of a stable base pair with C67, as previously proposed, but also another defect. A U6-U80G strain in which mispairing with position 67 is prevented grows poorly and assembles aberrant spliceosomes that retain U1 snRNP and fail to fully unwind the U4/U6 complex at elevated temperatures. Our data suggest that the U6 ISL bulge is important for coupling U1 snRNP release with U4/U6 unwinding during spliceosome activation.
Asunto(s)
ARN Nuclear Pequeño/química , ARN Nuclear Pequeño/genética , Saccharomyces cerevisiae/genética , Empalmosomas/metabolismo , Emparejamiento Base , Secuencia de Bases , Secuencia Conservada , Modelos Moleculares , Mutación , Conformación de Ácido Nucleico , Fenotipo , ARN de Hongos/química , ARN de Hongos/genética , Empalmosomas/genéticaRESUMEN
Most eukaryotic genes are transcribed by RNA polymerase II (Pol II), including those that produce mRNAs and many noncoding functional RNAs. Proper expression of these genes requires efficient termination by Pol II to avoid transcriptional interference and synthesis of extended, nonfunctional RNAs. We previously described a pathway for yeast Pol II termination that involves recognition of an element in the nascent transcript by the essential RNA-binding protein Nrd1. The Nrd1-dependent pathway appears to be used primarily for nonpolyadenylated transcripts, such as the small nuclear and small nucleolar RNAs (snoRNAs). mRNAs are thought to use a distinct pathway that is coupled to cleavage and polyadenylation of the transcript. Here we show that the terminator elements for two yeast snoRNA genes also direct polyadenylated 3'-end formation in the context of an mRNA 3' untranslated region. A selection for cis-acting terminator readthrough mutations identified conserved features of these elements, some of which are similar to cleavage and polyadenylation signals. A selection for trans-acting mutations that induce readthrough of both a snoRNA and an mRNA terminator yielded mutations in the Rpb3 and Rpb11 subunits of Pol II that define a remarkably discrete surface on the trailing end of the enzyme. Our results suggest that, at least in budding yeast, protein-coding and noncoding Pol II-transcribed genes use similar mechanisms to direct termination and that the termination signal is transduced through the Rpb3/Rpb11 heterodimer.