- Email: [email protected]
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Nuclear mRNA surveillance Shobha Vasudevan and Stuart W Peltzy Recent studies demonstrate that the factors involved in mRNA processing specify the fate of a transcript. The mRNA is either committed for export to the cytoplasm or accumulates in the nucleus, where it may be degraded. These studies reveal crosstalk among the nuclear events faced by the pre-mRNA. It is becoming evident that the components of the mRNA synthesis machinery interact with each other to establish a distinct surveillance mechanism that determines release of the transcript from the transcription site for further export and utilisation. Recent advances suggest that the major nuclear decay machinery, the nuclear exosome, and an Rrp6p-specific complex coordinate with processing factors to perform a unique regulatory function that determines its fate: either confinement of the defective mRNA at its transcription site, or release from its site of transcription for further processing and export or decay. Furthermore, message-specific regulatory mechanisms correspond with the nuclear mRNA synthesis machinery to control gene expression. Addresses Department of Molecular Genetics, Microbiology and Immunology, University of Medicine and Dentistry of New Jersey, Robert Wood Johnson Medical School and yCancer Institute of New Jersey, 675 Hoes Lane, Piscataway, NJ 08854, USA e-mail: [email protected]
Current Opinion in Cell Biology 2003, 15:332–337 This review comes from a themed issue on Nucleus and gene expression Edited by Jeanne Lawrence and Gordon Hager 0955-0674/03/$ – see front matter ß 2003 Elsevier Science Ltd. All rights reserved. DOI 10.1016/S0955-0674(03)00051-6
Abbreviations ARE AU-rich element NAS nonsense-associated altered splicing NMD nonsense-mediated decay pol II RNA polymerase II mRNP messenger ribonucleoprotein complex TREX transcription/export complex
Introduction mRNA synthesis involves a complex series of processing steps that determine the future of a transcript. Several lines of evidence suggest that these processes communicate and mutually influence the status of the transcript [1,2]. Development of the mature mRNA proceeds through a progressive sequence of interdependent events in the nucleus: transcription, capping, splicing, 30 -end Current Opinion in Cell Biology 2003, 15:332–337
cleavage, polyadenylation and mRNA release and export to the cytoplasm. Recent studies in yeast reveal that export factors, as well as mRNA processing factors, are recruited as a co-transcriptional complex. The export factors might themselves be required for termination of the processing events and subsequent release of the mRNA from the site of transcription [1,3,4,5]. These events also engage a quality-control measure that proofreads the transcript at each phase of production of the nascent mRNA. This validation of the transcript is essential for a newly uncovered step in mRNA synthesis: release of the transcript from the transcription site or unit [6,7,8,9]. Interestingly, the nonsense-mediated decay (NMD) pathway, a general surveillance mechanism, which involves recognition of nonsense codons as an aberration in the mRNA and prevents expression, also incorporates a nuclear proofing step that alters the splicing machinery and prevents release of specific transcripts [10,11]. A key component of the RNA processing machinery is the nuclear exosome, which comprises a versatile multicomponent complex of exonucleases and accessory factors [12–14]. Like its cytoplasmic counterpart, the nuclear exosome is a 30 !50 exonuclease complex; however, it is distinguished by a specific, nuclear-restricted exonuclease component, Rrp6p. This complex is required for small nuclear RNA (snRNA), ribosomal RNA (rRNA), small nucleolar RNA (snoRNA) and pre-mRNA maturation and for nuclear RNA degradation. Interestingly, the nuclear exosome, which is the major decay machinery for nuclear RNA, is involved in release of the transcript from the site of transcription. In addition to degrading premRNAs targeted for decay, Rrp6p plays a novel qualitycontrol role in monitoring the release of mRNA from the transcription site [8]. Modulation of Rrp6p or nuclear exosome functions can therefore lead to either accumulation of the aberrant transcripts at the site of transcription or can promote their processing and release for export. Transcripts that are processed aberrantly or slowly are targeted for decay by this unique surveillance mechanism [15,16,17]. Additionally, both the nuclear and cytoplasmic exosome can target AU-rich element (ARE)-bearing transcripts for decay, suggesting that the exosome is employed by message-specific regulatory systems to provide stringent control over expression of particular genes [18,19].
The transcription checkpoint Several processing and export factors are recruited along with RNA polymerase II (pol II) into the nascent mRNP (messenger ribonucleoprotein complex) [4,20]. Acquisition of the 7-methyl guanosine cap structure, followed by www.current-opinion.com
Nuclear mRNA surveillance Vasudevan and Peltz 333
Figure 1
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Quality-control surveillance of nuclear mRNA by an exosome/Rrp6p-dependent mechanism to ensure that accurately processed mRNA are exported or, alternatively, retained at the transcription site for exosome decay. mRNA synthesis involves a series of processing steps to synthesise mature transcript that is proofread in parallel by nuclear surveillance mechanisms at each step. The splicing (‘splicing’, green) and processing components (cleavage factors are represented as ‘cleavage’ [orange ovals] and the polyadenylation machinery as PAP [poly(A) polymerase], brown), as well as export factors (‘export’, red ovals) are co-transcriptionally recruited to the nascent transcript, possibly by the transcription complex (polII, purple) and the correct message forms the export-competent mRNP that is eventually exported and expressed in the cytoplasm. Alternatively, aberrant synthesis or processing leads to retention of the defective transcript at the site of transcription for further exosome-mediated decay. The cap is depicted as a red circle and Rrp6 is representative of nuclear surveillance mechanisms.
binding of the cap-binding complex (CBC), takes place co-transcriptionally on the growing mRNA [21–23]. The CBC is essential for both mRNA stability and splicing of the first intron, and its assembly is dependent on pol II [24–29]. Efficient export of mature mRNAs to the cytoplasm requires packaging into mRNP particles through accurate processing (Figure 1). Evidence for intricate interactions that connect transcription, processing and export have been found primarily in yeast but might also be conserved in mammalian systems [1,7,8,15,30,31]. Recent studies in yeast and HeLa cells show the existence of a conserved transcription, splicing and export complex — TREX (transcription/export complex) or THO–Sub2p–Yra1p complex [3,8,15,32]. This www.current-opinion.com
trimeric complex, which is recruited with pol II, demonstrates a critical association of Hpr1p, a member of the THO transcription elongation complex, with Sub2p, a key splicing factor, and with the mRNA export factor, Yra1p [3,8,15,32]. Yra1p binds Mex67p, the major exporter for mRNA. Yra1p has been shown to interact with Sub2p in yeast, and its mammalian homologues — ALY/REF — interacts with the Sub2p homologue, UAP56, in higher eukaryotes, linking splicing to export [32,33]. Interestingly, Yra1p recruitment to spliced transcripts requires accurate splicing and proper 30 -end formation for spliced and unspliced transcripts [2]. Disruption of yra1, sub2 or hpr1 results in nuclear retention and Current Opinion in Cell Biology 2003, 15:332–337
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transcript decay. These data suggest that accurate processing events, as well as co-transcriptional recruitment of the export factors, are essential for mRNA export (Figure 1). These factors show a strong genetic link with Rrp6p [8,15]. For example, deletion of Rrp6p in mutant strains of the components of TREX complex (yra1, sub2 or hpr1) alleviates the nuclear pre-mRNA accumulation, suggesting that Rrp6p and the nuclear exosome are involved in selecting the mRNA for nuclear accumulation and degradation or release for export.
Splicing and 30 -end formation Splicing and 30 -end formation reactions are required for mRNA export, presumably through the formation of export-competent mRNPs [1,2,31,34,35]. Cleavage mutants of the CFIA 30 -end processing complex, which is required to couple termination of transcription to cleavage and polyadenylation [36], were isolated in a yeast genetic screen for mutants defective in mRNA export, suggesting that these factors — or the processing reaction — is required to enable export [5]. However, correctly cleaved and polyadenylated transcripts also accumulate in the nucleus of cells expressing certain mutations of export and splicing factors — for example, rat7-1/nup159-1, yra1-1 or sub2-85 cells — suggesting a new function of such factors in the additional step of export-competent mRNP release, or alternatively, targeting the transcripts for accumulation and exosomemediated decay (Figure 1) [5,7,8,15]. The first evidence for a nuclear degradation pathway was the observation that an rrp6 mutation could suppress the pap1-1 temperature-sensitive mutation of poly(A) polymerase 1 [17]. Absence of the Rrp6p complex in the pap1-1 strain allowed for an increase in poly(A)þ mRNA levels by inactivating a nuclear degradation process and thereby permitting a residual polyadenylation of mRNA. Analysis of such transcripts revealed that although the abundance of the transcript did not change when Rrp6p was deleted, processed intermediates were apparently formed and could be translated, suggesting that overriding this Rrp6p function in transcript retention enabled the transcript to be processed and exported. A corollary of these observations was that there existed a competition between the 30 -end processing machinery and the Rrp6p-mediated degradation pathway. Alternatively, absence of the Rrp6p complex might enhance the processing of the mRNA [37]. Mutating components of the nuclear exosome in cells that are defective for splicing leads to accumulation of premRNAs, establishing the existence of a nuclear premRNA turnover pathway that competes with the splicing machinery to degrade aberrant, unspliced transcripts [38]. Interestingly, this proofing mechanism is regulated in yeast by the carbon source in which the yeast were grown. Glucose media was found to promote nuclear-exosomeCurrent Opinion in Cell Biology 2003, 15:332–337
mediated decay, whereas non-fermentable media inactivated exosome-dependent turnover of aberrant transcripts (although processing of other RNA by the nuclear exosome was maintained). Therefore, a component or associated factor of the nuclear exosome must be regulated by the carbon source. A recent study further delineated the existence of at least two distinct quality-control steps in this pre-mRNA degradation pathway. In the rna14-1 and the rna15-2 cleavage mutants, transcription termination and 30 -end formation are abrogated, leading to the production of 30 -extended transcripts. These transcripts were found to be degraded by the nuclear exosome into a decay intermediate that was further processed by a unique glucose-regulated mechanism to produce a population of translatable, heterogeneously polyadenylated transcripts [16]. Therefore, the fate of aberrant transcripts is regulated: in non-fermentable cultures, the transcripts are processed into functional mRNA species; glucose cultures favour degradation at this step. This additional step is reminiscent of the Rrp6p-dependent retention of inaccurately processed transcripts in a mechanism that competes with the processing machinery [17]. Intriguingly, the rna14-1 mutant was shown to require Rrp6p to promote decay at this step. Another study demonstrated that mRNAs from an rna14-3 cleavage-defective mutant, as well as a strain where transcripts are produced by ribozyme cleavage of the 30 end, prevent mRNA release at the transcription site in an Rrp6p-dependent mechanism [8]. Therefore, the 30 -end formation reactions are necessary for the production of an exportable mRNP. Retention of aberrant mRNPs requires Rrp6p. The accumulated transcript is then marked for either degradation or, in non-glucose cultures, for further processing (Figure 1). It remains to be investigated whether the proofreading and retention of nascent transcripts is a glucose-regulated mechanism and the components of this distinct step are yet to be identified.
mRNA release and export Transcripts accumulate as hyperadenylated mRNA at discrete transcription foci as a consequence of mutations in certain mRNA export factors (such as rat7-1 and rip1D) [7,39]. Other export mutants, such as xpo1-1 and mex67-5, which show strong processing defects, and yra1-1, which shows accurate cleavage and polyadenylation, also accumulate pre-mRNA at the transcription site, suggesting that mRNA export and processing reactions are interdependent with a common function that enables release of the nascent transcript [5]. When the nuclear exosome is inactivated, these foci become diffuse and the transcript increases in abundance. Furthermore, in certain exportdefective mutants, such as rat7-1, inactivation of the nuclear exosome permits export and translation [9]. The nuclear exosome component Rrp6p therefore defines a unique safeguard, where it controls release of www.current-opinion.com
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accurately processed transcripts for export or retention, and decay of the invalid transcript at the transcription site (Figure 1) [8]. Mutations in the mRNA export factor Npl3p block poly(A)þ mRNA export [40]. Npl3p is recruited only onto pol II transcripts and requires ongoing transcription. It also interacts with pol II and TATAbinding protein (TBP). This suggests that the decision to export the mRNA is made early on during transcription [41]. Significantly, Npl3p interacts with Rrp6p but not with other components of the exosome, and therefore might be involved in the Rrp6p-dependent accumulation of improperly processed mRNPs [17,37]. Furthermore, both proper 30 -end formation and splicing influence the release and export of the mature transcript by Yra1p in an Rrp6p-dependent mechanism [15]. Therefore, the nuclear-exosome-mediated proofing involves an additional Rrp6p-dependent step, which regulates the accumulation of improperly processed transcripts, as opposed to their release and export.
Message-specific targeting There is a growing literature identifying message-specific sequences that determine the fate of the transcript. One of the best-studied examples is Rev-mediated RRE (Rev recognition element) recognition, which overrides the nuclear proofreading mechanisms to allow Crm1-dependent export of unspliced genomic viral RNA for packaging [42–44]). An analogous system occurs for CTE (constitutive transport element)-containing transcripts that bind directly to the cellular mRNA export factor TAP (or NXF1 [nuclear RNA export factor 1]) to export intron-containing viral RNA [45–47]. Two post-transcriptional regulatory mechanisms, the NMD pathway and the ARE-mediated regulation, exert control at multiple stages of transcript maturation to prevent aberrant expression of specific transcripts. Premature termination codon recognition
NMD eliminates transcripts containing a premature nonsense codon present upstream of a ‘mark’ that targets these transcripts for decay [48–50]. This mark has been shown to bear the exon–EJC (exon junction complex), a composite of export and processing factors that fail to be removed by the translation ribosome and thereby invites decay of the transcript. This complex is loaded on by the spliceosome and promotes export of the matured mRNA. As the transcript matures and is exported and translated, the EJC or mark is remodelled. However, in the case of premature nonsense codons, the translating ribosome fails to remove this complex, which then serves to nucleate the NMD complex and targets the mRNA for decay. Recent studies have illustrated a new mechanism of regulation of nuclear transcripts by premature termination codons (PTCs) in transcripts encoding for the immunoglobulin M m gene or a derivative of the T cell receptor-b (TCR-b) gene [11,51]. Recognition of a nonsense www.current-opinion.com
codon by the NMD pathway appears to cause not only decay of such mRNAs but also modulates the splicing machinery to eliminate production of mature mRNA bearing the offending nonsense codon. The imperfect PTC-bearing transcript was also shown to cause an accumulation of the unspliced pre-mRNA at the transcription site, thereby reducing the production of the aberrant transcript [10]. This process, nonsense-associated altered splicing (NAS), requires decoding of the reading frame by an as yet unravelled mechanism that requires at least one of the factors involved in NMD [51]. Therefore, specific mRNAs experience yet another layer of proofing wherein the transcript reading frame is recognised to alter splicing and release of the inaccurate mRNA from its transcription site. AU-rich elements
AREs, well-studied stability and translation determinants [52–54], also regulate the localisation of transcripts in response to environmental stimuli [55,56]. A ubiquitous ARE-binding protein, HuR, has been demonstrated to not only stabilise ARE-bearing transcripts but also regulates the export and cytoplasmic localisations of such transcripts [57,58,59]. Under normal conditions, HuR, using transportin as its transporter, can export the AREbearing mRNA to the cytoplasm, which would allow expression of the transcripts. Under stress conditions, such as heat shock, however, HuR utilises Crm1 as its transporter, and acts as a selective export adaptor protein, exporting only Hsp70 and perhaps other such ARE-bearing stress-induced transcripts [60,61]. Although HuR is transported on heat shock, its association with poly(A)þ RNA is observed only in the nucleus, suggesting that HuR plays an additional stress-induced role of protecting ARE-bearing transcripts that accumulate in the nucleus upon stress conditions. Several proteins have been shown to bind AREs, and it is possible that HuR binds the ARE initially, presumably to protect the transcript from the exosome and export it to the cytoplasm for further translation. Interestingly, both the nuclear and cytoplasmic exosome can target ARE-bearing transcripts for decay [18,19], suggesting that the nuclear surveillance function of the exosome might be utilised to degrade ARE-bearing transcripts that do not form the export-competent mRNP complex under altered conditions.
Conclusions Recent advances connect the transcription elongation complex, processing, and export factors with the nuclear exosome in a common proofreading mechanism whereby inappropriately processed transcripts are retained and accumulate at the transcription site. It remains to be investigated whether this surveillance mechanism is invoked by a reading-frame surveillance pathway, NAS, to switch splicing and prevent release of specific messages. Additionally, the exosome surveillance mechanism might target ARE-containing transcripts for decay in Current Opinion in Cell Biology 2003, 15:332–337
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response to conditions that alter protective ARE-binding proteins. Intriguingly, Rrp6p modulates mRNA retention in certain processing and export mutants. This Rrp6pdependent checkpoint represents an additional level of regulation whereby in certain situations, such as nonfermentable carbon sources in yeast, accumulation and processing of the aberrantly synthesised transcript is favoured, whereas in glucose conditions the transcripts are targeted for degradation. Analogous situations may exist in higher eukaryotes where the nuclear exosome presents an additional layer of control that determines the fate of the nuclear transcript.
Acknowledgements We thank Carol J Wilusz and C Phillips for critical reading of the manuscript. This work was supported by a grant from the National Institutes of Health to SW Peltz (GM58276).
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Current Opinion in Cell Biology 2003, 15:332–337
Nuclear mRNA surveillance Shobha Vasudevan and Stuart W Peltzy Recent studies demonstrate that the factors involved in mRNA processing specify the fate of a transcript. The mRNA is either committed for export to the cytoplasm or accumulates in the nucleus, where it may be degraded. These studies reveal crosstalk among the nuclear events faced by the pre-mRNA. It is becoming evident that the components of the mRNA synthesis machinery interact with each other to establish a distinct surveillance mechanism that determines release of the transcript from the transcription site for further export and utilisation. Recent advances suggest that the major nuclear decay machinery, the nuclear exosome, and an Rrp6p-specific complex coordinate with processing factors to perform a unique regulatory function that determines its fate: either confinement of the defective mRNA at its transcription site, or release from its site of transcription for further processing and export or decay. Furthermore, message-specific regulatory mechanisms correspond with the nuclear mRNA synthesis machinery to control gene expression. Addresses Department of Molecular Genetics, Microbiology and Immunology, University of Medicine and Dentistry of New Jersey, Robert Wood Johnson Medical School and yCancer Institute of New Jersey, 675 Hoes Lane, Piscataway, NJ 08854, USA e-mail: [email protected]
Current Opinion in Cell Biology 2003, 15:332–337 This review comes from a themed issue on Nucleus and gene expression Edited by Jeanne Lawrence and Gordon Hager 0955-0674/03/$ – see front matter ß 2003 Elsevier Science Ltd. All rights reserved. DOI 10.1016/S0955-0674(03)00051-6
Abbreviations ARE AU-rich element NAS nonsense-associated altered splicing NMD nonsense-mediated decay pol II RNA polymerase II mRNP messenger ribonucleoprotein complex TREX transcription/export complex
Introduction mRNA synthesis involves a complex series of processing steps that determine the future of a transcript. Several lines of evidence suggest that these processes communicate and mutually influence the status of the transcript [1,2]. Development of the mature mRNA proceeds through a progressive sequence of interdependent events in the nucleus: transcription, capping, splicing, 30 -end Current Opinion in Cell Biology 2003, 15:332–337
cleavage, polyadenylation and mRNA release and export to the cytoplasm. Recent studies in yeast reveal that export factors, as well as mRNA processing factors, are recruited as a co-transcriptional complex. The export factors might themselves be required for termination of the processing events and subsequent release of the mRNA from the site of transcription [1,3,4,5]. These events also engage a quality-control measure that proofreads the transcript at each phase of production of the nascent mRNA. This validation of the transcript is essential for a newly uncovered step in mRNA synthesis: release of the transcript from the transcription site or unit [6,7,8,9]. Interestingly, the nonsense-mediated decay (NMD) pathway, a general surveillance mechanism, which involves recognition of nonsense codons as an aberration in the mRNA and prevents expression, also incorporates a nuclear proofing step that alters the splicing machinery and prevents release of specific transcripts [10,11]. A key component of the RNA processing machinery is the nuclear exosome, which comprises a versatile multicomponent complex of exonucleases and accessory factors [12–14]. Like its cytoplasmic counterpart, the nuclear exosome is a 30 !50 exonuclease complex; however, it is distinguished by a specific, nuclear-restricted exonuclease component, Rrp6p. This complex is required for small nuclear RNA (snRNA), ribosomal RNA (rRNA), small nucleolar RNA (snoRNA) and pre-mRNA maturation and for nuclear RNA degradation. Interestingly, the nuclear exosome, which is the major decay machinery for nuclear RNA, is involved in release of the transcript from the site of transcription. In addition to degrading premRNAs targeted for decay, Rrp6p plays a novel qualitycontrol role in monitoring the release of mRNA from the transcription site [8]. Modulation of Rrp6p or nuclear exosome functions can therefore lead to either accumulation of the aberrant transcripts at the site of transcription or can promote their processing and release for export. Transcripts that are processed aberrantly or slowly are targeted for decay by this unique surveillance mechanism [15,16,17]. Additionally, both the nuclear and cytoplasmic exosome can target AU-rich element (ARE)-bearing transcripts for decay, suggesting that the exosome is employed by message-specific regulatory systems to provide stringent control over expression of particular genes [18,19].
The transcription checkpoint Several processing and export factors are recruited along with RNA polymerase II (pol II) into the nascent mRNP (messenger ribonucleoprotein complex) [4,20]. Acquisition of the 7-methyl guanosine cap structure, followed by www.current-opinion.com
Nuclear mRNA surveillance Vasudevan and Peltz 333
Figure 1
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Quality-control surveillance of nuclear mRNA by an exosome/Rrp6p-dependent mechanism to ensure that accurately processed mRNA are exported or, alternatively, retained at the transcription site for exosome decay. mRNA synthesis involves a series of processing steps to synthesise mature transcript that is proofread in parallel by nuclear surveillance mechanisms at each step. The splicing (‘splicing’, green) and processing components (cleavage factors are represented as ‘cleavage’ [orange ovals] and the polyadenylation machinery as PAP [poly(A) polymerase], brown), as well as export factors (‘export’, red ovals) are co-transcriptionally recruited to the nascent transcript, possibly by the transcription complex (polII, purple) and the correct message forms the export-competent mRNP that is eventually exported and expressed in the cytoplasm. Alternatively, aberrant synthesis or processing leads to retention of the defective transcript at the site of transcription for further exosome-mediated decay. The cap is depicted as a red circle and Rrp6 is representative of nuclear surveillance mechanisms.
binding of the cap-binding complex (CBC), takes place co-transcriptionally on the growing mRNA [21–23]. The CBC is essential for both mRNA stability and splicing of the first intron, and its assembly is dependent on pol II [24–29]. Efficient export of mature mRNAs to the cytoplasm requires packaging into mRNP particles through accurate processing (Figure 1). Evidence for intricate interactions that connect transcription, processing and export have been found primarily in yeast but might also be conserved in mammalian systems [1,7,8,15,30,31]. Recent studies in yeast and HeLa cells show the existence of a conserved transcription, splicing and export complex — TREX (transcription/export complex) or THO–Sub2p–Yra1p complex [3,8,15,32]. This www.current-opinion.com
trimeric complex, which is recruited with pol II, demonstrates a critical association of Hpr1p, a member of the THO transcription elongation complex, with Sub2p, a key splicing factor, and with the mRNA export factor, Yra1p [3,8,15,32]. Yra1p binds Mex67p, the major exporter for mRNA. Yra1p has been shown to interact with Sub2p in yeast, and its mammalian homologues — ALY/REF — interacts with the Sub2p homologue, UAP56, in higher eukaryotes, linking splicing to export [32,33]. Interestingly, Yra1p recruitment to spliced transcripts requires accurate splicing and proper 30 -end formation for spliced and unspliced transcripts [2]. Disruption of yra1, sub2 or hpr1 results in nuclear retention and Current Opinion in Cell Biology 2003, 15:332–337
334 Nucleus and gene expression
transcript decay. These data suggest that accurate processing events, as well as co-transcriptional recruitment of the export factors, are essential for mRNA export (Figure 1). These factors show a strong genetic link with Rrp6p [8,15]. For example, deletion of Rrp6p in mutant strains of the components of TREX complex (yra1, sub2 or hpr1) alleviates the nuclear pre-mRNA accumulation, suggesting that Rrp6p and the nuclear exosome are involved in selecting the mRNA for nuclear accumulation and degradation or release for export.
Splicing and 30 -end formation Splicing and 30 -end formation reactions are required for mRNA export, presumably through the formation of export-competent mRNPs [1,2,31,34,35]. Cleavage mutants of the CFIA 30 -end processing complex, which is required to couple termination of transcription to cleavage and polyadenylation [36], were isolated in a yeast genetic screen for mutants defective in mRNA export, suggesting that these factors — or the processing reaction — is required to enable export [5]. However, correctly cleaved and polyadenylated transcripts also accumulate in the nucleus of cells expressing certain mutations of export and splicing factors — for example, rat7-1/nup159-1, yra1-1 or sub2-85 cells — suggesting a new function of such factors in the additional step of export-competent mRNP release, or alternatively, targeting the transcripts for accumulation and exosomemediated decay (Figure 1) [5,7,8,15]. The first evidence for a nuclear degradation pathway was the observation that an rrp6 mutation could suppress the pap1-1 temperature-sensitive mutation of poly(A) polymerase 1 [17]. Absence of the Rrp6p complex in the pap1-1 strain allowed for an increase in poly(A)þ mRNA levels by inactivating a nuclear degradation process and thereby permitting a residual polyadenylation of mRNA. Analysis of such transcripts revealed that although the abundance of the transcript did not change when Rrp6p was deleted, processed intermediates were apparently formed and could be translated, suggesting that overriding this Rrp6p function in transcript retention enabled the transcript to be processed and exported. A corollary of these observations was that there existed a competition between the 30 -end processing machinery and the Rrp6p-mediated degradation pathway. Alternatively, absence of the Rrp6p complex might enhance the processing of the mRNA [37]. Mutating components of the nuclear exosome in cells that are defective for splicing leads to accumulation of premRNAs, establishing the existence of a nuclear premRNA turnover pathway that competes with the splicing machinery to degrade aberrant, unspliced transcripts [38]. Interestingly, this proofing mechanism is regulated in yeast by the carbon source in which the yeast were grown. Glucose media was found to promote nuclear-exosomeCurrent Opinion in Cell Biology 2003, 15:332–337
mediated decay, whereas non-fermentable media inactivated exosome-dependent turnover of aberrant transcripts (although processing of other RNA by the nuclear exosome was maintained). Therefore, a component or associated factor of the nuclear exosome must be regulated by the carbon source. A recent study further delineated the existence of at least two distinct quality-control steps in this pre-mRNA degradation pathway. In the rna14-1 and the rna15-2 cleavage mutants, transcription termination and 30 -end formation are abrogated, leading to the production of 30 -extended transcripts. These transcripts were found to be degraded by the nuclear exosome into a decay intermediate that was further processed by a unique glucose-regulated mechanism to produce a population of translatable, heterogeneously polyadenylated transcripts [16]. Therefore, the fate of aberrant transcripts is regulated: in non-fermentable cultures, the transcripts are processed into functional mRNA species; glucose cultures favour degradation at this step. This additional step is reminiscent of the Rrp6p-dependent retention of inaccurately processed transcripts in a mechanism that competes with the processing machinery [17]. Intriguingly, the rna14-1 mutant was shown to require Rrp6p to promote decay at this step. Another study demonstrated that mRNAs from an rna14-3 cleavage-defective mutant, as well as a strain where transcripts are produced by ribozyme cleavage of the 30 end, prevent mRNA release at the transcription site in an Rrp6p-dependent mechanism [8]. Therefore, the 30 -end formation reactions are necessary for the production of an exportable mRNP. Retention of aberrant mRNPs requires Rrp6p. The accumulated transcript is then marked for either degradation or, in non-glucose cultures, for further processing (Figure 1). It remains to be investigated whether the proofreading and retention of nascent transcripts is a glucose-regulated mechanism and the components of this distinct step are yet to be identified.
mRNA release and export Transcripts accumulate as hyperadenylated mRNA at discrete transcription foci as a consequence of mutations in certain mRNA export factors (such as rat7-1 and rip1D) [7,39]. Other export mutants, such as xpo1-1 and mex67-5, which show strong processing defects, and yra1-1, which shows accurate cleavage and polyadenylation, also accumulate pre-mRNA at the transcription site, suggesting that mRNA export and processing reactions are interdependent with a common function that enables release of the nascent transcript [5]. When the nuclear exosome is inactivated, these foci become diffuse and the transcript increases in abundance. Furthermore, in certain exportdefective mutants, such as rat7-1, inactivation of the nuclear exosome permits export and translation [9]. The nuclear exosome component Rrp6p therefore defines a unique safeguard, where it controls release of www.current-opinion.com
Nuclear mRNA surveillance Vasudevan and Peltz 335
accurately processed transcripts for export or retention, and decay of the invalid transcript at the transcription site (Figure 1) [8]. Mutations in the mRNA export factor Npl3p block poly(A)þ mRNA export [40]. Npl3p is recruited only onto pol II transcripts and requires ongoing transcription. It also interacts with pol II and TATAbinding protein (TBP). This suggests that the decision to export the mRNA is made early on during transcription [41]. Significantly, Npl3p interacts with Rrp6p but not with other components of the exosome, and therefore might be involved in the Rrp6p-dependent accumulation of improperly processed mRNPs [17,37]. Furthermore, both proper 30 -end formation and splicing influence the release and export of the mature transcript by Yra1p in an Rrp6p-dependent mechanism [15]. Therefore, the nuclear-exosome-mediated proofing involves an additional Rrp6p-dependent step, which regulates the accumulation of improperly processed transcripts, as opposed to their release and export.
Message-specific targeting There is a growing literature identifying message-specific sequences that determine the fate of the transcript. One of the best-studied examples is Rev-mediated RRE (Rev recognition element) recognition, which overrides the nuclear proofreading mechanisms to allow Crm1-dependent export of unspliced genomic viral RNA for packaging [42–44]). An analogous system occurs for CTE (constitutive transport element)-containing transcripts that bind directly to the cellular mRNA export factor TAP (or NXF1 [nuclear RNA export factor 1]) to export intron-containing viral RNA [45–47]. Two post-transcriptional regulatory mechanisms, the NMD pathway and the ARE-mediated regulation, exert control at multiple stages of transcript maturation to prevent aberrant expression of specific transcripts. Premature termination codon recognition
NMD eliminates transcripts containing a premature nonsense codon present upstream of a ‘mark’ that targets these transcripts for decay [48–50]. This mark has been shown to bear the exon–EJC (exon junction complex), a composite of export and processing factors that fail to be removed by the translation ribosome and thereby invites decay of the transcript. This complex is loaded on by the spliceosome and promotes export of the matured mRNA. As the transcript matures and is exported and translated, the EJC or mark is remodelled. However, in the case of premature nonsense codons, the translating ribosome fails to remove this complex, which then serves to nucleate the NMD complex and targets the mRNA for decay. Recent studies have illustrated a new mechanism of regulation of nuclear transcripts by premature termination codons (PTCs) in transcripts encoding for the immunoglobulin M m gene or a derivative of the T cell receptor-b (TCR-b) gene [11,51]. Recognition of a nonsense www.current-opinion.com
codon by the NMD pathway appears to cause not only decay of such mRNAs but also modulates the splicing machinery to eliminate production of mature mRNA bearing the offending nonsense codon. The imperfect PTC-bearing transcript was also shown to cause an accumulation of the unspliced pre-mRNA at the transcription site, thereby reducing the production of the aberrant transcript [10]. This process, nonsense-associated altered splicing (NAS), requires decoding of the reading frame by an as yet unravelled mechanism that requires at least one of the factors involved in NMD [51]. Therefore, specific mRNAs experience yet another layer of proofing wherein the transcript reading frame is recognised to alter splicing and release of the inaccurate mRNA from its transcription site. AU-rich elements
AREs, well-studied stability and translation determinants [52–54], also regulate the localisation of transcripts in response to environmental stimuli [55,56]. A ubiquitous ARE-binding protein, HuR, has been demonstrated to not only stabilise ARE-bearing transcripts but also regulates the export and cytoplasmic localisations of such transcripts [57,58,59]. Under normal conditions, HuR, using transportin as its transporter, can export the AREbearing mRNA to the cytoplasm, which would allow expression of the transcripts. Under stress conditions, such as heat shock, however, HuR utilises Crm1 as its transporter, and acts as a selective export adaptor protein, exporting only Hsp70 and perhaps other such ARE-bearing stress-induced transcripts [60,61]. Although HuR is transported on heat shock, its association with poly(A)þ RNA is observed only in the nucleus, suggesting that HuR plays an additional stress-induced role of protecting ARE-bearing transcripts that accumulate in the nucleus upon stress conditions. Several proteins have been shown to bind AREs, and it is possible that HuR binds the ARE initially, presumably to protect the transcript from the exosome and export it to the cytoplasm for further translation. Interestingly, both the nuclear and cytoplasmic exosome can target ARE-bearing transcripts for decay [18,19], suggesting that the nuclear surveillance function of the exosome might be utilised to degrade ARE-bearing transcripts that do not form the export-competent mRNP complex under altered conditions.
Conclusions Recent advances connect the transcription elongation complex, processing, and export factors with the nuclear exosome in a common proofreading mechanism whereby inappropriately processed transcripts are retained and accumulate at the transcription site. It remains to be investigated whether this surveillance mechanism is invoked by a reading-frame surveillance pathway, NAS, to switch splicing and prevent release of specific messages. Additionally, the exosome surveillance mechanism might target ARE-containing transcripts for decay in Current Opinion in Cell Biology 2003, 15:332–337
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response to conditions that alter protective ARE-binding proteins. Intriguingly, Rrp6p modulates mRNA retention in certain processing and export mutants. This Rrp6pdependent checkpoint represents an additional level of regulation whereby in certain situations, such as nonfermentable carbon sources in yeast, accumulation and processing of the aberrantly synthesised transcript is favoured, whereas in glucose conditions the transcripts are targeted for degradation. Analogous situations may exist in higher eukaryotes where the nuclear exosome presents an additional layer of control that determines the fate of the nuclear transcript.
Acknowledgements We thank Carol J Wilusz and C Phillips for critical reading of the manuscript. This work was supported by a grant from the National Institutes of Health to SW Peltz (GM58276).
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Current Opinion in Cell Biology 2003, 15:332–337