RESUMEN
From yeast to humans, pre-mRNA splicing occurs mainly co-transcriptionally, with splicing and transcription functionally coupled such that they influence one another. The recruitment model of co-transcriptional splicing proposes that core members of the transcription elongation machinery have the potential to influence co-transcriptional spliceosome assembly and pre-mRNA splicing. Here, we tested whether the transcription elongation kinases Bur1 and Ctk1 affect co-transcriptional spliceosome assembly and pre-mRNA splicing in the budding yeast Saccharomyces cerevisiae. In S. cerevisiae, Ctk1 is the major kinase that phosphorylates serine 2 of the carboxy-terminal domain of the largest subunit of RNA polymerase II, whilst Bur1 augments the kinase activity of Ctk1 and is the major kinase for elongation factor Spt5. We used the auxin-inducible degron system to conditionally deplete Bur1 and Ctk1 kinases, and investigated the effects on co-transcriptional spliceosome assembly and pre-mRNA splicing. Depletion of Ctk1 effectively reduced phosphorylation of serine 2 of the carboxy-terminal domain but did not impact co-transcriptional spliceosome assembly or pre-mRNA splicing. In striking contrast, depletion of Bur1 did not reduce phosphorylation of serine 2 of the carboxy-terminal domain, but reduced Spt5 phosphorylation and enhanced co-transcriptional spliceosome assembly and pre-mRNA splicing, suggesting a role for this kinase in modulating co-transcriptional splicing.
Asunto(s)
Quinasas Ciclina-Dependientes/metabolismo , Regulación Fúngica de la Expresión Génica , Proteínas Quinasas/metabolismo , Precursores del ARN/genética , Empalme del ARN , Proteínas de Saccharomyces cerevisiae/metabolismo , Empalmosomas/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Fosforilación , Factores de Elongación Transcripcional/metabolismoRESUMEN
The bloodstream form of Trypanosoma brucei persists in mammalian hosts through a population survival strategy depending on antigenic variation of a cell surface coat composed of the variant surface glycoprotein (VSG). The integrity of the VSG coat is essential and blocking its synthesis results in a cell division cycle arrest just prior to cytokinesis. This observation indicates that VSG levels are monitored and that the cell has mechanisms to respond to a disruption of synthesis. Here, the regulation of VSG mRNA levels has been investigated by first measuring VSG mRNA copy number, and second using ectopic expression of VSG transgenes containing premature termination codons. The findings are that (i) VSG mRNA copy number varies with the identity of the VSG and (ii) a pathway detects synthesis of non-functional VSG protein and results in an increase in VSG mRNA levels.
Asunto(s)
Regulación de la Expresión Génica , Trypanosoma brucei brucei/fisiología , Tripanosomiasis Africana/parasitología , Glicoproteínas Variantes de Superficie de Trypanosoma/genética , Línea Celular , Interacciones Huésped-Parásitos , ARN Mensajero/genética , Tripanosomiasis Africana/metabolismo , Glicoproteínas Variantes de Superficie de Trypanosoma/metabolismoRESUMEN
Pre-messenger RNA splicing involves multi-step assembly of the large spliceosome complexes that catalyse the two consecutive trans-esterification reactions, resulting in intron removal. There is evidence that proof-reading mechanisms monitor the fidelity of this complex process. Transcripts that fail these fidelity tests are thought to be directed to degradation pathways, permitting the splicing factors to be recycled. While studying the roles of splicing factors in vivo, in budding yeast, we performed targeted depletion of individual proteins, and analysed the effect on co-transcriptional spliceosome assembly and splicing efficiency. Unexpectedly, depleting factors such as Prp16 or Prp22, that are known to function at the second catalytic step or later in the splicing pathway, resulted in a defect in the first step of splicing, and accumulation of arrested spliceosomes. Through a kinetic analysis of newly synthesized RNA, we observed that a second step splicing defect (the primary defect) was rapidly followed by the first step of splicing defect. Our results show that knocking down a splicing factor can quickly lead to a recycling defect with splicing factors sequestered in stalled complexes, thereby limiting new rounds of splicing. We demonstrate that this 'feed-back' effect can be minimized by depleting the target protein more gradually or only partially, allowing a better separation between primary and secondary effects. Our findings indicate that splicing surveillance mechanisms may not always cope with spliceosome assembly defects, and suggest that work involving knock-down of splicing factors or components of other large complexes should be carefully monitored to avoid potentially misleading conclusions.
Asunto(s)
Retroalimentación Fisiológica/efectos de los fármacos , Empalme del ARN/efectos de los fármacos , ARN de Hongos/genética , ARN Mensajero/genética , Saccharomyces cerevisiae/efectos de los fármacos , Empalmosomas/efectos de los fármacos , Adenosina Trifosfatasas/deficiencia , Adenosina Trifosfatasas/genética , ARN Helicasas DEAD-box/deficiencia , ARN Helicasas DEAD-box/genética , Ácidos Indolacéticos/farmacología , División del ARN/efectos de los fármacos , ARN Helicasas/deficiencia , ARN Helicasas/genética , Precursores del ARN/genética , Precursores del ARN/metabolismo , Factores de Empalme de ARN/deficiencia , Factores de Empalme de ARN/genética , ARN de Hongos/metabolismo , ARN Mensajero/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Empalmosomas/genética , Empalmosomas/metabolismoRESUMEN
The plant auxin binding receptor, TIR1, recognizes proteins containing a specific auxin-inducible degron (AID) motif in the presence of auxin, targeting them for degradation. This system is exploited in many non-plant eukaryotes, such that a target protein, tagged with the AID motif, is degraded upon auxin addition. The level of TIR1 expression is critical; excessive expression leads to degradation of the AID-tagged protein even in the absence of auxin, whereas low expression leads to slow depletion. A ß-estradiol-inducible AID system was created, with expression of TIR1 under the control of a ß-estradiol inducible promoter. The level of TIR1 is tunable by changing the time of incubation with ß-estradiol before auxin addition. This protocol describes how to rapidly deplete a target protein using the AID system. The appropriate ß-estradiol incubation time depends on the abundance of the target protein. Therefore, efficient depletion depends on optimal timing that also minimizes auxin-independent depletion.
Asunto(s)
Proteínas de Plantas/metabolismo , Proteolisis , Receptores de Superficie Celular/metabolismo , Estradiol , Ácidos Indolacéticos/metabolismo , Transporte de ProteínasRESUMEN
There is increasing evidence from yeast to humans that pre-mRNA splicing occurs mainly cotranscriptionally, such that splicing and transcription are functionally coupled. Currently, there is little insight into the contribution of the core transcription elongation machinery to cotranscriptional spliceosome assembly and pre-mRNA splicing. Spt5 is a member of the core transcription elongation machinery and an essential protein, whose absence in budding yeast causes defects in pre-mRNA splicing. To determine how Spt5 affects pre-mRNA splicing, we used the auxin-inducible degron system to conditionally deplete Spt5 in Saccharomyces cerevisiae and assayed effects on cotranscriptional spliceosome assembly and splicing. We show that Spt5 is needed for efficient splicing and for the accumulation of U5 snRNPs at intron-containing genes, and therefore for stable cotranscriptional assembly of spliceosomes. The defect in cotranscriptional spliceosome assembly can explain the relatively mild splicing defect as being a consequence of the failure of cotranscriptional splicing. Coimmunoprecipitation of Spt5 with core spliceosomal proteins and all spliceosomal snRNAs suggests a model whereby Spt5 promotes cotranscriptional pre-mRNA splicing by stabilizing the association of U5 snRNP with spliceosome complexes as they assemble on the nascent transcript. If this phenomenon is conserved in higher eukaryotes, it has the potential to be important for cotranscriptional regulation of alternative splicing.
Asunto(s)
Proteínas Cromosómicas no Histona/fisiología , Saccharomyces cerevisiae/metabolismo , Empalmosomas , Transcripción Genética , Factores de Elongación Transcripcional/fisiología , Proteínas Cromosómicas no Histona/metabolismo , Inmunoprecipitación , Unión Proteica , Empalme del ARN , Ribonucleoproteína Nuclear Pequeña U5/genética , Factores de Elongación Transcripcional/metabolismoRESUMEN
The auxin-inducible degron (AID) is a useful technique to rapidly deplete proteins of interest in nonplant eukaryotes. Depletion is achieved by addition of the plant hormone auxin to the cell culture, which allows the auxin-binding receptor, TIR1, to target the AID-tagged protein for degradation by the proteasome. Fast depletion of the target protein requires good expression of TIR1 protein, but as we show here, high levels of TIR1 may cause uncontrolled depletion of the target protein in the absence of auxin. To enable conditional expression of TIR1 to a high level when required, we regulated the expression of TIR1 using the ß-estradiol expression system. This is a fast-acting gene induction system that does not cause secondary effects on yeast cell metabolism. We demonstrate that combining the AID and ß-estradiol systems results in a tightly controlled and fast auxin-induced depletion of nuclear target proteins. Moreover, we show that depletion rate can be tuned by modulating the duration of ß-estradiol preincubation. We conclude that TIR1 protein is a rate-limiting factor for target protein depletion in yeast, and we provide new tools that allow tightly controlled, tuneable, and efficient depletion of essential proteins whereas minimising secondary effects.
Asunto(s)
Proteínas Fúngicas/genética , Regulación Fúngica de la Expresión Génica , Proteínas de Choque Térmico/genética , Ácidos Indolacéticos/metabolismo , Proteínas Nucleares/genética , Saccharomycetales/genética , Estradiol , Expresión Génica , Transporte de Proteínas , Proteolisis , Saccharomycetales/metabolismo , Activación TranscripcionalRESUMEN
It is widely accepted that heterochromatin is necessary to maintain genomic stability. However, direct experimental evidence supporting this is slim. Previous studies using either enzyme inhibitors, gene knockout or knockdown studies all are subject to the caveat that drugs may have off-target effects and enzymes that modify chromatin proteins to support heterochromatin formation may also have numerous other cellular targets as well. Here, we describe PREditOR (protein reading and editing of residues), a synthetic biology approach that allows us to directly remove heterochromatin from cells without either drugs or global interference with gene function. We find that removal of heterochromatin perturbs mitotic progression and causes a dramatic increase in chromosome segregation defects, possibly as a result of interfering with the normal centromeric localization of the chromosomal passenger complex.