RESUMEN
Caenorhabditis elegans mutants deleted for TDP-1, an ortholog of the neurodegeneration-associated RNA-binding protein TDP-43, display only mild phenotypes. Nevertheless, transcriptome sequencing revealed that many RNAs were altered in accumulation and/or processing in the mutant. Analysis of these transcriptional abnormalities demonstrates that a primary function of TDP-1 is to limit formation or stability of double-stranded RNA. Specifically, we found that deletion of tdp-1: (1) preferentially alters the accumulation of RNAs with inherent double-stranded structure (dsRNA); (2) increases the accumulation of nuclear dsRNA foci; (3) enhances the frequency of adenosine-to-inosine RNA editing; and (4) dramatically increases the amount of transcripts immunoprecipitable with a dsRNA-specific antibody, including intronic sequences, RNAs with antisense overlap to another transcript, and transposons. We also show that TDP-43 knockdown in human cells results in accumulation of dsRNA, indicating that suppression of dsRNA is a conserved function of TDP-43 in mammals. Altered accumulation of structured RNA may account for some of the previously described molecular phenotypes (e.g., altered splicing) resulting from reduction of TDP-43 function.
Asunto(s)
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/fisiología , Proteínas de Unión al ADN/metabolismo , Estabilidad del ARN , ARN Bicatenario/metabolismo , Proteínas de Unión al ARN/metabolismo , Animales , Proteínas de Caenorhabditis elegans/genética , Proteínas de Unión al ADN/genética , Eliminación de Gen , Perfilación de la Expresión Génica , Humanos , Proteínas de Unión al ARN/genéticaRESUMEN
Spliced leader (SL) trans-splicing in Caenorhabditis elegans attaches a 22-nucleotide (nt) exon onto the 5' end of many mRNAs. A particular class of SL, SL2, splices mRNAs of downstream operon genes. Here we use an embryonic extract-based in vitro splicing system to show that SL2 specificity information is encoded within the polycistronic pre-mRNA, and that trans-splicing specificity is recapitulated in vitro. We define an RNA sequence required for SL2 trans-splicing, the U-rich (Ur) element, through mutational analysis and bioinformatics as a short stem-loop followed by a sequence motif, UAYYUU, located approximately 50 nt upstream of the trans-splice site. Furthermore, this element is predicted in intercistronic regions of numerous operons of C. elegans and other species that use SL2 trans-splicing. We propose that the UAYYUU motif hybridizes with the 5' splice site on the SL2 RNA to recruit the SL to the pre-mRNA. In this way, the UAYYUU motif in the pre-mRNA would serve an analogous function to the similar sequence in the U1 snRNA, which binds to the 5' splice site of introns, effectively reversing the roles of snRNP and pre-mRNA in trans-splicing.
Asunto(s)
Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Precursores del ARN/metabolismo , ARN Lider Empalmado/metabolismo , Ribonucleoproteínas Nucleares Pequeñas/metabolismo , Trans-Empalme , Animales , Secuencia de Bases/genética , Biología Computacional , Secuencia de Consenso/genética , Secuencias Invertidas Repetidas/genética , Precursores del ARN/química , Precursores del ARN/genética , ARN Lider Empalmado/genética , Ribonucleoproteínas Nucleares Pequeñas/química , Ribonucleoproteínas Nucleares Pequeñas/genética , Uridina/genéticaRESUMEN
More than half of Caenorhabditis elegans pre-mRNAs lose their original 5' ends in a process termed "trans-splicing" in which the RNA extending from the transcription start site (TSS) to the site of trans-splicing of the primary transcript, termed the "outron," is replaced with a 22-nt spliced leader. This complicates the mapping of TSSs, leading to a lack of available TSS mapping data for these genes. We used growth at low temperature and nuclear isolation to enrich for transcripts still containing outrons, applying a modified SAGE capture procedure and high-throughput sequencing to characterize 5' termini in this transcript population. We report from this data both a landscape of 5'-end utilization for C. elegans and a representative collection of TSSs for 7351 trans-spliced genes. TSS distributions for individual genes were often dispersed, with a greater average number of TSSs for trans-spliced genes, suggesting that trans-splicing may remove selective pressure for a single TSS. Upstream of newly defined TSSs, we observed well-known motifs (including TATAA-box and SP1) as well as novel motifs. Several of these motifs showed association with tissue-specific expression and/or conservation among six worm species. Comparing TSS features between trans-spliced and non-trans-spliced genes, we found stronger signals among outron TSSs for preferentially positioning of flanking nucleosomes and for downstream Pol II enrichment. Our data provide an enabling resource for both experimental and theoretical analysis of gene structure and function in C. elegans.
Asunto(s)
Caenorhabditis elegans/genética , Genes de Helminto , Sitio de Iniciación de la Transcripción , Regiones no Traducidas 5' , Animales , Secuencia de Bases , Secuencia Conservada , Regulación de la Expresión Génica , Anotación de Secuencia Molecular , Especificidad de Órganos , Regiones Promotoras Genéticas , ARN de Helminto/genética , ARN de Helminto/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Análisis de Secuencia de ADN , Trans-EmpalmeRESUMEN
In the United States, estimates indicate there are between 250,000 and 400,000 individuals with Down syndrome (DS), and nearly all will develop Alzheimer's disease (AD) pathology starting in their 30s. With the current lifespan being 55 to 60 years, approximately 70% will develop dementia, and if their life expectancy continues to increase, the number of individuals developing AD will concomitantly increase. Pathogenic and mechanistic links between DS and Alzheimer's prompted the Alzheimer's Association to partner with the Linda Crnic Institute for Down Syndrome and the Global Down Syndrome Foundation at a workshop of AD and DS experts to discuss similarities and differences, challenges, and future directions for this field. The workshop articulated a set of research priorities: (1) target identification and drug development, (2) clinical and pathological staging, (3) cognitive assessment and clinical trials, and (4) partnerships and collaborations with the ultimate goal to deliver effective disease-modifying treatments.
Asunto(s)
Enfermedad de Alzheimer/fisiopatología , Síndrome de Down/fisiopatología , Enfermedad de Alzheimer/diagnóstico , Enfermedad de Alzheimer/tratamiento farmacológico , Enfermedad de Alzheimer/patología , Animales , Ensayos Clínicos como Asunto , Congresos como Asunto , Modelos Animales de Enfermedad , Síndrome de Down/diagnóstico , Síndrome de Down/tratamiento farmacológico , Síndrome de Down/patología , Descubrimiento de Drogas , Humanos , Pruebas NeuropsicológicasRESUMEN
Trans-splicing of one of two short leader RNAs, SL1 or SL2, occurs at the 5' ends of pre-mRNAs of many C. elegans genes. We have exploited RNA-sequencing data from the modENCODE project to analyze the transcriptome of C. elegans for patterns of trans-splicing. Transcripts of â¼70% of genes are trans-spliced, similar to earlier estimates based on analysis of far fewer genes. The mRNAs of most trans-spliced genes are spliced to either SL1 or SL2, but most genes are not trans-spliced to both, indicating that SL1 and SL2 trans-splicing use different underlying mechanisms. SL2 trans-splicing occurs in order to separate the products of genes in operons genome wide. Shorter intercistronic distance is associated with greater use of SL2. Finally, increased use of SL1 trans-splicing to downstream operon genes can indicate the presence of an extra promoter in the intercistronic region, creating what has been termed a "hybrid" operon. Within hybrid operons the presence of the two promoters results in the use of the two SL classes: Transcription that originates at the promoter upstream of another gene creates a polycistronic pre-mRNA that receives SL2, whereas transcription that originates at the internal promoter creates transcripts that receive SL1. Overall, our data demonstrate that >17% of all C. elegans genes are in operons.
Asunto(s)
Caenorhabditis elegans/genética , Trans-Empalme/genética , Animales , ADN Intergénico/genética , Eliminación de Gen , Masculino , Anotación de Secuencia Molecular , Operón/genética , Regiones Promotoras Genéticas , Precursores del ARN/genética , Precursores del ARN/metabolismo , Sitios de Empalme de ARN/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Lider Empalmado/genética , ARN Lider Empalmado/metabolismoRESUMEN
In Caenorhabditis elegans, the transcripts of many genes are trans-spliced to an SL1 spliced leader, a process that removes the RNA extending from the transcription start site to the trans-splice site, thereby making it difficult to determine the position of the promoter. Here we use RT-PCR to identify promoters of trans-spliced genes. Many genes in C. elegans are organized in operons where genes are closely clustered, typically separated by only ~100 nucleotides, and transcribed by an upstream promoter. The transcripts of downstream genes are trans-spliced to an SL2 spliced leader. The polycistronic precursor RNA is processed into individual transcripts by 3' end formation and trans-splicing. Although the SL2 spliced leader does not appear to be used for other gene arrangements, there is a relatively small number of genes whose transcripts are processed by SL2 but are not close to another gene in the same orientation. Although these genes do not appear to be members of classical C. elegans operons, we investigated whether these might represent unusual operons with long spacing or a different, nonoperon mechanism for specifying SL2 trans-splicing. We show transcription of the entire region between the SL2 trans-spliced gene and the next upstream gene, sometimes several kilobases distant, suggesting that these represent exceptional operons. We also report a second type of atypical "alternative" operon, in which 3' end formation and trans-splicing by SL2 occur within an intron. In this case, the processing sometimes results in a single transcript, and sometimes in two separate mRNAs.
Asunto(s)
Caenorhabditis elegans/genética , ADN Recombinante/genética , Operón/genética , Trans-Empalme/genética , Sitio de Iniciación de la Transcripción , Animales , Caenorhabditis elegans/metabolismo , Genoma de los Helmintos , Empalme del ARN/genética , ARN de Helminto/genética , ARN de Helminto/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Lider Empalmado/genética , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
The soil nematodes Caenorhabditis briggsae and Caenorhabditis elegans diverged from a common ancestor roughly 100 million years ago and yet are almost indistinguishable by eye. They have the same chromosome number and genome sizes, and they occupy the same ecological niche. To explore the basis for this striking conservation of structure and function, we have sequenced the C. briggsae genome to a high-quality draft stage and compared it to the finished C. elegans sequence. We predict approximately 19,500 protein-coding genes in the C. briggsae genome, roughly the same as in C. elegans. Of these, 12,200 have clear C. elegans orthologs, a further 6,500 have one or more clearly detectable C. elegans homologs, and approximately 800 C. briggsae genes have no detectable matches in C. elegans. Almost all of the noncoding RNAs (ncRNAs) known are shared between the two species. The two genomes exhibit extensive colinearity, and the rate of divergence appears to be higher in the chromosomal arms than in the centers. Operons, a distinctive feature of C. elegans, are highly conserved in C. briggsae, with the arrangement of genes being preserved in 96% of cases. The difference in size between the C. briggsae (estimated at approximately 104 Mbp) and C. elegans (100.3 Mbp) genomes is almost entirely due to repetitive sequence, which accounts for 22.4% of the C. briggsae genome in contrast to 16.5% of the C. elegans genome. Few, if any, repeat families are shared, suggesting that most were acquired after the two species diverged or are undergoing rapid evolution. Coclustering the C. elegans and C. briggsae proteins reveals 2,169 protein families of two or more members. Most of these are shared between the two species, but some appear to be expanding or contracting, and there seem to be as many as several hundred novel C. briggsae gene families. The C. briggsae draft sequence will greatly improve the annotation of the C. elegans genome. Based on similarity to C. briggsae, we found strong evidence for 1,300 new C. elegans genes. In addition, comparisons of the two genomes will help to understand the evolutionary forces that mold nematode genomes.
Asunto(s)
Caenorhabditis elegans/genética , Caenorhabditis/genética , Genoma , Genómica/métodos , Animales , Evolución Biológica , Mapeo Cromosómico , Cromosomas Artificiales Bacterianos , Análisis por Conglomerados , Codón , Secuencia Conservada , Evolución Molecular , Exones , Biblioteca de Genes , Secuencias Repetitivas Esparcidas , Intrones , MicroARNs/genética , Modelos Genéticos , Modelos Estadísticos , Datos de Secuencia Molecular , Familia de Multigenes , Sistemas de Lectura Abierta , Mapeo Físico de Cromosoma , Plásmidos/metabolismo , Estructura Terciaria de Proteína , Proteínas/química , ARN/química , ARN Ribosómico/genética , ARN Lider Empalmado , ARN de Transferencia/genética , Análisis de Secuencia de ADN , Especificidad de la EspecieRESUMEN
Trisomy 21 (T21) causes Down syndrome (DS), but the mechanisms by which T21 produces the different disease spectrum observed in people with DS are unknown. We recently identified an activated interferon response associated with T21 in human cells of different origins, consistent with overexpression of the four interferon receptors encoded on chromosome 21, and proposed that DS could be understood partially as an interferonopathy. However, the impact of T21 on systemic signaling cascades in living individuals with DS is undefined. To address this knowledge gap, we employed proteomics approaches to analyze blood samples from 263 individuals, 165 of them with DS, leading to the identification of dozens of proteins that are consistently deregulated by T21. Most prominent among these proteins are numerous factors involved in immune control, the complement cascade, and growth factor signaling. Importantly, people with DS display higher levels of many pro-inflammatory cytokines (e.g. IL-6, MCP-1, IL-22, TNF-α) and pronounced complement consumption, resembling changes seen in type I interferonopathies and other autoinflammatory conditions. Therefore, these results are consistent with the hypothesis that increased interferon signaling caused by T21 leads to chronic immune dysregulation, and justify investigations to define the therapeutic value of immune-modulatory strategies in DS.
Asunto(s)
Síndrome de Down/sangre , Inflamación/sangre , Proteoma/análisis , Adolescente , Adulto , Niño , Preescolar , Enfermedad Crónica , Proteínas del Sistema Complemento/análisis , Citocinas/sangre , Síndrome de Down/complicaciones , Femenino , Humanos , Lactante , Inflamación/complicaciones , Masculino , Receptores de Factores de Crecimiento/sangre , Trisomía , Adulto JovenRESUMEN
UAP56 is an essential eukaryotic pre-mRNA splicing factor and mRNA export factor. The mechanisms of its functions are not well understood. We determined the crystal structures of the N- and C-terminal domains of human UAP56 (comprising 90% of the full-length UAP56) at 1.9 A resolution. The two domains each have a RecA-like fold and are connected by a flexible linker. The overall fold of each domain is highly similar to the corresponding domains of eIF4A (a prototypic DExD/H-box protein), with differences at the loops and termini. This structural similarity suggests that UAP56 is likely to possess ATPase and helicase activity similar to eIF4A. The NTP binding pocket of UAP56 is occupied by a citrate ion, mimicking the phosphates of NTP and retaining the P loop in an open conformation. The crystal structure of the N-terminal domain of UAP56 also reveals a dimer interface that is potentially important for UAP56's function.
Asunto(s)
Factor 4A Eucariótico de Iniciación/química , Modelos Moleculares , ARN Helicasas/química , Empalme del ARN/genética , Transporte de ARN/fisiología , Secuencia de Aminoácidos , Sitios de Unión , Cristalografía por Rayos X , ARN Helicasas DEAD-box , Humanos , Datos de Secuencia Molecular , Estructura Terciaria de Proteína , Homología de Secuencia de AminoácidoRESUMEN
Transcription termination is mechanistically coupled to pre-mRNA 3' end formation to prevent transcription much beyond the gene 3' end. C. elegans, however, engages in polycistronic transcription of operons in which 3' end formation between genes is not accompanied by termination. We have performed RNA polymerase II (RNAPII) and CstF ChIP-seq experiments to investigate at a genome-wide level how RNAPII can transcribe through multiple poly-A signals without causing termination. Our data shows that transcription proceeds in some ways as if operons were composed of multiple adjacent single genes. Total RNAPII shows a small peak at the promoter of the gene cluster and a much larger peak at 3' ends. These 3' peaks coincide with maximal phosphorylation of Ser2 within the C-terminal domain (CTD) of RNAPII and maximal localization of the 3' end formation factor CstF. This pattern occurs at all 3' ends including those at internal sites in operons where termination does not occur. Thus the normal mechanism of 3' end formation does not always result in transcription termination. Furthermore, reduction of CstF50 by RNAi did not substantially alter the pattern of CstF64, total RNAPII, or Ser2 phosphorylation at either internal or terminal 3' ends. However, CstF50 RNAi did result in a subtle reduction of CstF64 binding upstream of the site of 3' cleavage, suggesting that the CstF50/CTD interaction may facilitate bringing the 3' end machinery to the transcription complex.
Asunto(s)
Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/genética , Factor de Estimulación del Desdoblamiento/metabolismo , ARN Polimerasa II/metabolismo , Transcripción Genética , Animales , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Genes de Helminto , Operón , Fosforilación , Regiones Promotoras Genéticas , Serina/metabolismoRESUMEN
Metazoan introns contain a polypyrimidine tract immediately upstream of the AG dinucleotide that defines the 3' splice site. In the nematode Caenorhabditis elegans, 3' splice sites are characterized by a highly conserved UUUUCAG/R octamer motif. While the conservation of pyrimidines in this motif is strongly suggestive of their importance in pre-mRNA splicing, in vivo evidence in support of this is lacking. In an N-ethyl-N-nitrosourea (ENU) mutagenesis screen in Caenorhabditis elegans, we have isolated a strain containing a point mutation in the octamer motif of a 3' splice site in the daf-12 gene. This mutation, a single base T-to-G transversion at the -5 position relative to the splice site, causes a strong daf-12 loss-of-function phenotype by abrogating splicing. The resulting transcript is predicted to encode a truncated DAF-12 protein generated by translation into the retained intron, which contains an in-frame stop codon. Other than the perfectly conserved AG dinucleotide at the site of splicing, G at the -5 position of the octamer motif is the most uncommon base in C. elegans 3' splice sites, occurring at closely paired sites where the better match to the splicing consensus is a few bases downstream. Our results highlight both the biological importance of the highly conserved -5 uridine residue in the C. elegans 3' splice site octamer motif as well as the utility of using ENU as a mutagen to study the function of polypyrimidine tracts and other AU- or AT-rich motifs in vivo.
Asunto(s)
Caenorhabditis elegans/efectos de los fármacos , Caenorhabditis elegans/genética , Etilnitrosourea/toxicidad , Intrones , Mutagénesis/efectos de los fármacos , Sitios de Empalme de ARN , Animales , Secuencia de Bases , Caenorhabditis elegans/metabolismo , Mapeo Cromosómico , Mutación , Motivos de Nucleótidos , Fenotipo , Polimorfismo de Nucleótido Simple , Unión Proteica , Factores de Empalme de ARN/metabolismo , Eliminación de SecuenciaRESUMEN
Nearly 15% of the ~20,000 C. elegans genes are contained in operons, multigene clusters controlled by a single promoter. The vast majority of these are of a type where the genes in the cluster are ~100 bp apart and the pre-mRNA is processed by 3' end formation accompanied by trans-splicing. A spliced leader, SL2, is specialized for operon processing. Here we summarize current knowledge on several variations on this theme including: (1) hybrid operons, which have additional promoters between genes; (2) operons with exceptionally long (> 1 kb) intercistronic regions; (3) operons with a second 3' end formation site close to the trans-splice site; (4) alternative operons, in which the exons are sometimes spliced as a single gene and sometimes as two genes; (5) SL1-type operons, which use SL1 instead of SL2 to trans-splice and in which there is no intercistronic space; (6) operons that make dicistronic mRNAs; and (7) non-operon gene clusters, in which either two genes use a single exon as the 3' end of one and the 5' end of the next, or the 3' UTR of one gene serves as the outron of the next. Each of these variations is relatively infrequent, but together they show a remarkable variety of tight-linkage gene arrangements in the C. elegans genome.
Asunto(s)
Caenorhabditis elegans/genética , Genes de Helminto , Familia de Multigenes , Operón , AnimalesRESUMEN
About 70% of C. elegans mRNAs are trans-spliced to one of two 22 nucleotide spliced leaders. SL1 is used to trim off the 5' ends of pre-mRNAs and replace them with the SL1 sequence. This processing event is very closely related to cis-splicing, or intron removal. The SL1 sequence is donated by a 100 nt small nuclear ribonucleoprotein particle (snRNP), the SL1 snRNP. This snRNP is structurally and functionally similar to the U snRNAs (U1, U2, U4, U5 and U6) that play key roles in intron removal and trans-splicing, except that the SL1 snRNP is consumed in the process. More than half of C. elegans pre-mRNAs are subject to SL1 trans-splicing, whereas ~30% are not trans-spliced. The remaining genes are trans-spliced by SL2, which is donated by a similar snRNP, the SL2 snRNP. SL2 recipients are all downstream genes in closely spaced gene clusters similar to bacterial operons. They are transcribed from a promoter at the 5' end of the cluster of between 2 and 8 genes. This transcription makes a polycistronic pre-mRNA that is co-transcriptionally processed by cleavage and polyadenylation at the 3' end of each gene, and this event is closely coupled to the SL2 trans-splicing event that occurs only ~100 nt further downstream. SL2 trans-splicing requires a sequence between the genes, the Ur element, that likely base pairs with the 5' splice site on the SL2 snRNP, in a manner analogous to the interaction between the 5' splice site in cis-splicing with the U1 snRNP. The key difference is that in trans-splicing, the snRNP contains the 5' splice site, whereas in cis-splicing the pre-mRNA does. Some operons, termed "hybrid operons", contain an additional promoter between two genes that can express the downstream gene or genes with a developmental profile that is different from that of the entire operon. The operons contain primarily genes required for rapid growth, including genes whose products are needed for mitochondrial function and the basic machinery of gene expression. Recent evidence suggests that RNA polymerase is poised at the promoters of growth genes, and operons allow more efficient recovery from growth-arrested states, resulting in reduction in the need for this cache of inactive RNA polymerase.
Asunto(s)
Caenorhabditis elegans/genética , Operón , Trans-Empalme , Animales , Evolución Molecular , Genes de Helminto , ARN de Helminto , Ribonucleoproteínas Nucleares PequeñasRESUMEN
Some genes in the candidate early-branching eukaryote Giardia lamblia occur in separate pieces, transcribed from non-contiguous chromosomal locations. The pre-mRNAs from the separate pieces apparently find each other by regions of complementarity and are subsequently spliced together by the spliceosome. Could genes in pieces, transcribed into separate pre-mRNAs, have been an early feature of spliceosomal evolution?
Asunto(s)
Giardia lamblia/genética , Empalme del ARN , ADN Protozoario/genética , Regulación de la Expresión Génica/fisiología , Empalme de Proteína , Proteínas Protozoarias , Sitios de Empalme de ARN , ARN Mensajero/genética , ARN Protozoario/genética , Empalmosomas/metabolismoRESUMEN
Trans-splicing is the joining together of portions of two separate pre-mRNA molecules. The two distinct categories of spliceosomal trans-splicing are genic trans-splicing, which joins exons of different pre-mRNA transcripts, and spliced leader (SL) trans-splicing, which involves an exon donated from a specialized SL RNA. Both depend primarily on the same signals and components as cis-splicing. Genic trans-splicing events producing protein-coding mRNAs have been described in a variety of organisms, including Caenorhabditis elegans and Drosophila. In mammalian cells, genic trans-splicing can be associated with cancers and translocations. SL trans-splicing has mainly been studied in nematodes and trypanosomes, but there are now numerous and diverse phyla (including primitive chordates) where this type of trans-splicing has been detected. Such diversity raises questions as to the evolutionary origin of the process. Another intriguing question concerns the function of trans-splicing, as operon resolution can only account for a small proportion of the total amount of SL trans-splicing.
Asunto(s)
Trans-Empalme/genética , Trans-Empalme/fisiología , Animales , Secuencia de Bases , Evolución Molecular , Modelos Biológicos , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Operón , Filogenia , Precursores del ARN/genética , Precursores del ARN/metabolismo , Estabilidad del ARN , ARN Lider Empalmado/química , ARN Lider Empalmado/genética , ARN Lider Empalmado/metabolismo , Empalmosomas/metabolismoRESUMEN
In Caenorhabditis elegans, newly transcribed RNA is processed in several novel ways. Although introns are removed by a canonical spliceosome, they have evolved several specialized features that reflect the differences in the way they are recognized and the way they are spliced. C. elegans introns are unusually short, in part because they have no specific branch-point sequences and contain minimal polypryimidine tracts. Instead, their 3' splice site is characterized by a highly conserved consensus sequence, which alone may be sufficient to position all spliceosomal elements at the 3' end of the intron. Many RNA molecules are also trans-spliced: a capped 22nt RNA leader is donated by one of a family of specialized snRNPs and spliced to an unpaired 3' splice site, usually just upstream of the start codon. The RNA upstream of this splice site, the outron, is removed during trans-splicing and presumably degraded, making the identification of the transcriptional start site problematic. Transcripts from approximately 70% of all genes are trans-spliced. Trans-splicing has enabled the evolution of operons - multigene clusters in which a single upstream promoter drives the transcription of a polycistronic pre-mRNA. The C. elegans genome contains more than 1000 such operons. The polycistronic pre-mRNA is processed into individual gene-encoding mRNAs by coordinated upstream 3' end formation and downstream trans-splicing. An intercistronic RNA sequence, the Ur element, plays a key role in specifying downstream trans-splicing.
Asunto(s)
Caenorhabditis elegans/genética , Procesamiento Postranscripcional del ARN , ARN de Helminto/metabolismo , Animales , Secuencia de Bases , Caenorhabditis elegans/metabolismo , Componentes del Gen , Genes de Helminto , Datos de Secuencia Molecular , Operón , Sitios de Empalme de ARNRESUMEN
The heptad repeat of the RNA polymerase II (RNAPII) C-terminal domain is phosphorylated at serine 5 near gene 5' ends and serine 2 near 3' ends in order to recruit pre-mRNA processing factors. Ser-5(P) is associated with gene 5' ends to recruit capping enzymes, whereas Ser-2(P) is associated with gene 3' ends to recruit cleavage and polyadenylation factors. In the gene clusters called operons in Caenorhabditis elegans, there is generally only a single promoter, but each gene in the operon forms a 3' end by the usual mechanism. Although downstream operon genes have 5' ends, they receive their caps by trans splicing rather than by capping enzymes. Thus, they are predicted to not need Ser-5 phosphorylation. Here we show by RNAPII chromatin immunoprecipitation (ChIP) that internal operon gene 5' ends do indeed lack Ser-5(P) peaks. In contrast, Ser-2(P) peaks occur at each mRNA 3' end, where the 3'-end formation machinery binds. These results provide additional support for the idea that the serine phosphorylation of the C-terminal domain (CTD) serves to bring RNA-processing enzymes to the transcription complex. Furthermore, these results provide a novel demonstration that genes in operons are cotranscribed from a single upstream promoter.
Asunto(s)
Caenorhabditis elegans/genética , Genes , ARN Polimerasa II/metabolismo , Animales , Caenorhabditis elegans/metabolismo , Operón , Fosforilación , ARN Polimerasa II/genética , Precursores del ARN/genética , Precursores del ARN/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Trans-Empalme , Factores de Escisión y Poliadenilación de ARNm/genética , Factores de Escisión y Poliadenilación de ARNm/metabolismoRESUMEN
Many mRNAs in Caenorhabditis elegans are generated through a trans-splicing reaction that adds one of two classes of spliced leader RNA to an independently transcribed pre-mRNA. SL1 leaders are spliced mostly to pre-mRNAs from genes with outrons, intron-like sequences at the 5'-ends of the pre-mRNAs. In contrast, SL2 leaders are nearly exclusively trans-spliced to genes that occur downstream in polycistronic pre-mRNAs produced from operons. Operon pre-mRNA processing requires separation into individual transcripts, which is accomplished by 3'-processing of upstream genes and spliced leader trans-splicing to the downstream genes. We used a novel computational analysis, based on nonnegative matrix factorization, to identify and characterize significant differences in the cis-acting sequence elements that differentiate various types of functional site, including internal versus terminal 3'-processing sites, and SL1 versus SL2 trans-splicing sites. We describe several key elements, including the U-rich (Ur) element that couples 3'-processing with SL2 trans-splicing, and a novel outron (Ou) element that occurs upstream of SL1 trans-splicing sites. Finally, we present models of the distinct classes of trans-splicing reaction, including SL1 trans-splicing at the outron, SL2 trans-splicing in standard operons, competitive SL1-SL2 trans-splicing in operons with large intergenic separation, and SL1 trans-splicing in SL1-type operons, which have no intergenic separation.
Asunto(s)
Caenorhabditis elegans/genética , Operón/fisiología , Precursores del ARN/metabolismo , Procesamiento Postranscripcional del ARN/fisiología , Sitios de Empalme de ARN/genética , ARN de Helminto/metabolismo , Trans-Empalme/fisiología , Animales , Caenorhabditis elegans/química , Caenorhabditis elegans/fisiología , Modelos Genéticos , Precursores del ARN/genética , ARN de Helminto/genética , ARN Lider Empalmado/genética , ARN Lider Empalmado/metabolismoRESUMEN
In Caenorhabditis elegans, the small nuclear ribonucleoprotein (snRNP)-associated proteins U1A and U2B'' are approximately 50% identical to each other, and neither bears signature characteristics of mammalian U1A or U2B'' or the single Drosophila homolog, SNF. We show here that the genes that encode these proteins (rnp-2 and rnp-3) are cotranscribed in an operon, and that ribonucleoprotein RNP-2 is U1 snRNP-associated (U1A) whereas RNP-3 is U2 snRNP-associated (U2B''). U2B'' interacts with U2 even in the absence of another U2 snRNP protein, U2A'. Like U1A and U2B'' from yeast, plants, and vertebrates, worm U1A and U2B'' are more similar to each other than they are to other U1A or U2B'' proteins, respectively. Even though U1A and U2B'' interact with different snRNPs, they are functionally redundant; knockout of both is required for a lethal phenotype. Interestingly, U1A associates with U2 RNA when U2B'' is deleted. Thus, the two members of this gene family normally function as components of different snRNPs but apparently remain capable of performing the function of the other. Redundancy results from the fact that one protein can substitute for the other, even though it normally does not.