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Nuclear messenger RNA (mRNA) export is vital for cell survival under both physiological and stress conditions. To cope with stress, cells block bulk mRNA export while selectively exporting stress-specific mRNAs. Under physiological conditions, nuclear adaptor proteins recruit the mRNA exporter to the mRNA for export. By contrast, during stress conditions, the mRNA exporter is likely directly recruited to stress-specific mRNAs at their transcription sites to facilitate selective mRNA export. In this review, we summarize our current understanding of nuclear mRNA export. Importantly, we explore insights into the mechanisms that block bulk mRNA export and facilitate transcript-specific mRNA export under stress, highlighting the gaps that still need to be filled., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

The Prp19 complex (Prp19C), also named NineTeen Complex (NTC), is conserved from yeast to human and functions in many different processes such as genome stability, splicing, and transcription elongation. In the latter, Prp19C ensures TREX occupancy at transcribed genes. TREX, in turn, couples transcription to nuclear mRNA export by recruiting the mRNA exporter to transcribed genes and consequently to nascent mRNAs. Here, we assess the function of the nonessential Prp19C subunit Syf2 and the nonessential Prp19C-associated protein Cwc15 in the interaction of Prp19C and TREX with the transcription machinery, Prp19C and TREX occupancy, and transcription elongation. Whereas both proteins are important for Prp19C-TREX interaction, Syf2 is needed for full Prp19C occupancy, and Cwc15 is important for the interaction of Prp19C with RNA polymerase II and TREX occupancy. These partially overlapping functions are corroborated by a genetic interaction between Δcwc15 and Δsyf2 Finally, Cwc15 also interacts genetically with the transcription elongation factor Dst1 and functions in transcription elongation. In summary, we uncover novel roles of the Prp19C component Syf2 and the Prp19C-associated protein Cwc15 in Prp19C's function in transcription elongation., (© 2024 Henke-Schulz et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

The conserved TREX complex has multiple functions in gene expression such as transcription elongation, 3' end processing, mRNP assembly and nuclear mRNA export as well as the maintenance of genomic stability. In Saccharomyces cerevisiae , TREX is composed of the pentameric THO complex, the DEAD-box RNA helicase Sub2, the nuclear mRNA export adaptor Yra1, and the SR-like proteins Gbp2 and Hrb1. Here, we present the structural analysis of the endogenous TREX complex of S. cerevisiae purified from its native environment. To this end, we used cross-linking mass spectrometry to gain structural information on regions of the complex that are not accessible to classical structural biology techniques. We also used negative-stain electron microscopy to investigate the organization of the cross-linked complex used for XL-MS by comparing our endogenous TREX complex with recently published structural models of recombinant THO-Sub2 complexes. According to our analysis, the endogenous yeast TREX complex preferentially assembles into a dimer., (© 2023 Kern et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

RNA-binding proteins (RBPs) control every RNA metabolic process by multiple protein-RNA and protein-protein interactions. Their roles have largely been analyzed by crude mutations, which abrogate multiple functions at once and likely impact the structural integrity of the large ribonucleoprotein particles (RNPs) these proteins function in. Using UV-induced RNA-protein crosslinking of entire cells, protein complex purification and mass spectrometric analysis, we identified >100 in vivo RNA crosslinks in 16 nuclear mRNP components in Saccharomyces cerevisiae. For functional analysis, we chose Npl3, which displayed crosslinks in its two RNA recognition motifs (RRMs) and in the connecting flexible linker region. Both RRM domains and the linker uniquely contribute to RNA recognition as revealed by NMR and structural analyses. Interestingly, mutations in these regions cause different phenotypes, indicating distinct functions of the different RNA-binding domains. Notably, an npl3-Linker mutation strongly impairs recruitment of several mRNP components to chromatin and incorporation of other mRNP components into nuclear mRNPs, establishing a so far unknown function of Npl3 in nuclear mRNP assembly. Taken together, our integrative analysis uncovers a specific function of the RNA-binding activity of the nuclear mRNP component Npl3. This approach can be readily applied to RBPs in any RNA metabolic process., (© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Signal transduction and the regulation of gene expression are fundamental processes in every cell. RNA-binding proteins (RBPs) play a key role in the post-transcriptional modulation of gene expression in response to both internal and external stimuli. However, how signaling pathways regulate the assembly of RBPs with mRNAs remains largely unknown. Here, we summarize observations showing that the formation and composition of messenger ribonucleoprotein particles (mRNPs) is dynamically remodeled in space and time by specific signaling cascades and the resulting post-translational modifications. The integration of signaling events with gene expression is key to the rapid adaptation of cells to environmental changes and stress. Only a combined approach analyzing the signal transduction pathways and the changes in post-transcriptional gene expression they cause will unravel the mechanisms coordinating these important cellular processes.

Hundreds of canonical RNA binding proteins facilitate diverse and essential RNA processing steps in cells forming a central regulatory point in gene expression. However, recent discoveries including the identification of a large number of noncanonical proteins bound to RNA have changed our view on RNA-protein interactions merely as necessary steps in RNA biogenesis. As the list of proteins interacting with RNA has expanded, so has the scope of regulation through RNA-protein interactions. In addition to facilitating RNA metabolism, RNA binding proteins help to form subcellular structures and membraneless organelles, and provide means to recruit components of macromolecular complexes to their sites of action. Moreover, RNA-protein interactions are not static in cells but the ribonucleoprotein (RNP) complexes are highly dynamic in response to cellular cues. The identification of novel proteins in complex with RNA and ways cells use these interactions to control cellular functions continues to broaden the scope of RNA regulation in cells and the current challenge is to move from cataloguing the components of RNPs into assigning them functions. This will not only facilitate our understanding of cellular homeostasis but may bring in key insights into human disease conditions where RNP components play a central role. This review brings together the classical view of regulation accomplished through RNA-protein interactions with the novel insights gained from the identification of RNA binding interactomes. We discuss the challenges in combining molecular mechanism with cellular functions on the journey towards a comprehensive understanding of the regulatory functions of RNA-protein interactions in cells. This article is categorized under: RNA Interactions with Proteins and Other Molecules > Protein-RNA Interactions: Functional Implications aRNA Interactions with Proteins and Other Molecules > RNA-Protein Complexes RNA Interactions with Proteins and Other Molecules > Protein-RNA Recognition., (© 2019 Wiley Periodicals, Inc.)

mRNA is the "hermes" of gene expression as it carries the information of a protein-coding gene to the ribosome. Already during its synthesis, the mRNA is bound by mRNA-binding proteins that package the mRNA into a messenger ribonucleoprotein particle (mRNP). This mRNP assembly is important for mRNA stability and nuclear mRNA export. It also often regulates later steps in the mRNA lifetime such as translation and mRNA degradation in the cytoplasm. Thus, mRNP composition and accordingly the assembly of nuclear mRNA-binding proteins onto the mRNA are of crucial importance for correct gene expression. Here, we review our current knowledge of the mechanism of co-transcriptional mRNP assembly and nuclear mRNA export. We introduce the proteins involved and elaborate on what is known about their functions so far. In addition, we discuss the importance of regulated mRNP assembly in changing environmental conditions, especially during stress. Furthermore, we examine how defects in mRNP assembly cause diseases and how viruses exploit the host's nuclear mRNA export pathway. Finally, we summarize the questions that need to be answered in the future.

The different steps of gene expression are intimately linked to coordinate and regulate this complex process. During transcription, numerous RNA-binding proteins are already loaded onto the nascent mRNA and package the mRNA into a messenger ribonucleoprotein particle (mRNP). These RNA-binding proteins are often also involved in other steps of gene expression than mRNA packaging. For example, TREX functions in transcription, mRNP packaging and nuclear mRNA export. Previously, we showed that the Prp19 splicing complex (Prp19C) is needed for efficient transcription as well as TREX occupancy at transcribed genes. Here, we show that the splicing factor Mud2 interacts with Prp19C and is needed for Prp19C occupancy at transcribed genes in Saccharomyces cerevisiae. Interestingly, Mud2 is not only recruited to intron-containing but also to intronless genes indicating a role in transcription. Indeed, we show for the first time that Mud2 functions in transcription. Furthermore, these functions of Mud2 are likely evolutionarily conserved as Mud2 is also recruited to an intronless gene and interacts with Prp19C in Drosophila melanogaster. Taken together, we classify Mud2 as a novel transcription factor that is necessary for the recruitment of mRNA-binding proteins to the transcription machinery. Thus, Mud2 is a multifunctional protein important for transcription, splicing and most likely also mRNP packaging.

RNA polymerase III (RNAPIII) synthesizes diverse, small, non-coding RNAs with many important roles in the cellular metabolism. One of the open questions of RNAPIII transcription is whether and how additional factors are involved. Recently, Nab2 was identified as the first messenger ribonucleoprotein particle (mRNP) biogenesis factor with a function in RNAPIII transcription.

RNA polymerase III (RNAPIII) synthesizes most small RNAs, the most prominent being tRNAs. Although the basic mechanism of RNAPIII transcription is well understood, recent evidence suggests that additional proteins play a role in RNAPIII transcription. Here, we discovered by a genome-wide approach that Nab2, a poly(A)-binding protein important for correct poly(A) tail length and nuclear mRNA export, is present at all RNAPIII transcribed genes. The occupancy of Nab2 at RNAPIII transcribed genes is dependent on transcription. Using a novel temperature-sensitive allele of NAB2, nab2-34, we show that Nab2 is required for the occupancy of RNAPIII and TFIIIB at target genes. Furthermore, Nab2 interacts with RNAPIII, TFIIIB, and RNAPIII transcripts. Importantly, impairment of Nab2 function causes an RNAPIII transcription defect in vivo and in vitro. Taken together, we establish Nab2, an important mRNA biogenesis factor, as a novel player required for RNAPIII transcription by stabilizing TFIIIB and RNAPIII at promoters., (© 2015 Reuter et al.; Published by Cold Spring Harbor Laboratory Press.)

In eukaryotes, the messenger RNA (mRNA), the blueprint of a protein-coding gene, is processed and packaged into a messenger ribonucleoprotein particle (mRNP) by mRNA-binding proteins in the nucleus. The steps of mRNP formation - transcription, processing, packaging, and the orchestrated release of the export-competent mRNP from the site of transcription for nuclear mRNA export - are tightly coupled to ensure a highly efficient and regulated process. The importance of highly accurate nuclear mRNP formation is illustrated by the fact that mutations in components of this pathway lead to cellular inviability or to severe diseases in metazoans. We hypothesize that efficient mRNP formation is realized by a molecular mRNP packaging station, which is built by several recruitment platforms and coordinates the individual steps of mRNP formation., (© 2015 The Authors. Bioessays published by WILEY Periodicals, Inc.)

Translation is a fundamental and highly regulated cellular process. Previously, we reported that the kinase and transcription elongation factor Ctk1 increases fidelity during translation elongation in Saccharomyces cerevisiae. Here, we show that loss of Ctk1 function also affects the initiation step of translation. Translation active extracts from Ctk1-depleted cells show impaired translation activity of capped mRNA, but not mRNA reporters containing the cricket paralysis virus (CrPV) internal ribosome entry site (IRES). Furthermore, the formation of 80S initiation complexes is decreased, which is probably due to reduced subunit joining. In addition, we determined the changes in the phosphorylation pattern of a ribosome enriched fraction after depletion of Ctk1. Thus, we provide a catalogue of phosphoproteomic changes dependent on Ctk1. Taken together, our data suggest a stimulatory function of Ctk1 in 80S formation during translation initiation., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Transcription elongation is a highly dynamic and discontinuous process, which includes frequent pausing of RNA polymerase II (RNAPII). RNAPII complexes that stall persistently on a gene during transcription elongation block transcription and thus have to be removed. It has been proposed that the cellular pathway for removal of these DNA damage-independently stalled RNAPII complexes is similar or identical to the removal of RNAPII complexes stalled due to DNA damage. Here, we show that-consistent with previous data-DNA damage-independent stalling causes polyubiquitylation and proteasome-mediated degradation of Rpb1, the largest subunit of RNAPII, using Saccharomyces cerevisiae as model system. Moreover, recruitment of the proteasome to RNAPII and transcribed genes is increased when transcription elongation is impaired indicating that Rpb1 degradation takes place at the gene. Importantly, in contrast to the DNA damage-dependent pathway Rpb1 degradation of DNA damage-independently stalled RNAPII is independent of the E3 ligase Elc1. In addition, deubiquitylation of RNAPII is also independent of the Elc1-antagonizing deubiquitylase Ubp3. Thus, the pathway for degradation of DNA damage-independently stalled RNAPII is overlapping yet distinct from the previously described pathway for degradation of RNAPII stalled due to DNA damage. Taken together, we provide the first evidence that the cell discriminates between DNA damage-dependently and -independently stalled RNAPII., (© The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Messenger RNA (mRNA) synthesis and export are tightly linked, but the molecular mechanisms of this coupling are largely unknown. In Saccharomyces cerevisiae, the conserved TREX complex couples transcription to mRNA export and mediates mRNP formation. Here, we show that TREX is recruited to the transcription machinery by direct interaction of its subcomplex THO with the serine 2-serine 5 (S2/S5) diphosphorylated CTD of RNA polymerase II. S2 and/or tyrosine 1 (Y1) phosphorylation of the CTD is required for TREX occupancy in vivo, establishing a second interaction platform necessary for TREX recruitment in addition to RNA. Genome-wide analyses show that the occupancy of THO and the TREX components Sub2 and Yra1 increases from the 5' to the 3' end of the gene in accordance with the CTD S2 phosphorylation pattern. Importantly, in a mutant strain, in which TREX is recruited to genes but does not increase towards the 3' end, the expression of long transcripts is specifically impaired. Thus, we show for the first time that a 5'-3' increase of a protein complex is essential for correct expression of the genome. In summary, we provide insight into how the phospho-code of the CTD directs mRNP formation and export through TREX recruitment., Competing Interests: The authors have declared that no competing interests exist.

The conserved Prp19 complex (Prp19C) - also known as NineTeen Complex (NTC) - functions in several processes of paramount importance for cellular homeostasis. NTC/Prp19C was discovered as a complex that functions in splicing and more specifically during the catalytic activation of the spliceosome. More recent work revealed that NTC/Prp19C plays a role in transcription elongation in Saccharomyces cerevisiae and in genome maintenance in higher eukaryotes. In addition, mouse PRP19 might ubiquity late proteins targeted for degradation and guide them to the proteasome. Furthermore, NTC/Prp19C has been implicated in lipid droplet biogenesis. In the future, the molecular function of NTC/Prp19C in all of these processes needs to be refined or elucidated. Most of NTC/Prp19C's functions have been shown in only one or few organisms. However, since this complex is highly conserved it is likely that it has the same functions across all species. Moreover, one NTC/Prp19C or different subcomplexes could function in the above-mentioned processes. Intriguingly, NTC/Prp19C might link these different processes to ensure an optimal coordination of cellular processes. Thus, many important questions about the functions of this interesting complex remain to be investigated. In this review we discuss the different functions of NTC/Prp19C focusing on the novel and emerging ones as well as open questions., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Ribosome biogenesis is a process required for cellular growth and proliferation. Processing of ribosomal RNA (rRNA) is highly sensitive to flavopiridol, a specific inhibitor of cyclin-dependent kinase 9 (Cdk9). Cdk9 has been characterized as the catalytic subunit of the positive transcription elongation factor b (P-TEFb) of RNA polymerase II (RNAPII). Here we studied the connection between RNAPII transcription and rRNA processing. We show that inhibition of RNAPII activity by α-amanitin specifically blocks processing of rRNA. The block is characterized by accumulation of 3' extended unprocessed 47 S rRNAs and the entire inhibition of other 47 S rRNA-specific processing steps. The transcription rate of rRNA is moderately reduced after inhibition of Cdk9, suggesting that defective 3' processing of rRNA negatively feeds back on RNAPI transcription. Knockdown of Cdk9 caused a strong reduction of the levels of RNAPII-transcribed U8 small nucleolar RNA, which is essential for 3' rRNA processing in mammalian cells. Our data demonstrate a pivotal role of Cdk9 activity for coupling of RNAPII transcription with small nucleolar RNA production and rRNA processing.

The multiprotein complex Mediator is a coactivator of RNA polymerase (Pol) II transcription that is required for the regulated expression of protein-coding genes. Mediator serves as an end point of signaling pathways and regulates Pol II transcription, but the mechanisms it uses are not well understood. Here, we used mass spectrometry and dynamic transcriptome analysis to investigate a functional role of Mediator phosphorylation in gene expression. Affinity purification and mass spectrometry revealed that Mediator from the yeast Saccharomyces cerevisiae is phosphorylated at multiple sites of 17 of its 25 subunits. Mediator phosphorylation levels change upon an external stimulus set by exposure of cells to high salt concentrations. Phosphorylated sites in the Mediator tail subunit Med15 are required for suppression of stress-induced changes in gene expression under non-stress conditions. Thus dynamic and differential Mediator phosphorylation contributes to gene regulation in eukaryotic cells.

The Mre11-Rad50-Nbs1 (MRN) complex tethers, processes and signals DNA double-strand breaks, promoting genomic stability. To understand the functional architecture of MRN, we determined the crystal structures of the Schizosaccharomyces pombe Mre11 dimeric catalytic domain alone and in complex with a fragment of Nbs1. Two Nbs1 subunits stretch around the outside of the nuclease domains of Mre11, with one subunit additionally bridging and locking the Mre11 dimer via a highly conserved asymmetrical binding motif. Our results show that Mre11 forms a flexible dimer and suggest that Nbs1 not only is a checkpoint adaptor but also functionally influences Mre11-Rad50. Clinical mutations in Mre11 are located along the Nbs1-interaction sites and weaken the Mre11-Nbs1 interaction. However, they differentially affect DNA repair and telomere maintenance in Saccharomyces cerevisiae, potentially providing insight into their different human disease pathologies.

The La-motif (LAM) is an ancient and ubiquitous RNA-binding domain defining a superfamily of proteins, which comprises the genuine La proteins and La-related proteins (LARPs). In contrast to La, which binds and stabilizes pre-tRNAs and other RNA polymerase III transcripts, data on function and RNA targets of the LARPs have remained scarce. We have undertaken a global approach to elucidate the previously suggested role of the yeast LARP Slf1p in copper homeostasis. By applying RNA-binding protein immunopurification-microarray (RIP-Chip) analysis, we show that Slf1p and its paralog Sro9p copurify with overlapping sets of hundreds of functionally related mRNAs, including many transcripts coding for ribosomal proteins and histones. Interestingly, among these potential RNA targets were also mRNAs coding for proteins critical for protection of cells against elevated copper concentrations. Mutations introduced in the conserved aromatic patch of the LAM in Slf1p drastically impaired both association with its targets and Slf1-mediated protection of cells against toxic copper concentrations. Furthermore, we show that Slf1p stabilizes copper-related mRNA targets in a LAM-dependent manner. These results provide the first evidence for post-transcriptional regulation of factors/pathways implicated in copper homeostasis by a cytoplasmic RBP.

During transcription of protein coding genes by RNA Polymerase II the mRNA is processed and packaged into an mRNP. Among the proteins binding cotranscriptionally to the mRNP are mRNA export factors. One of the protein complexes thus coupling transcription to mRNA export is the TREX complex. However, despite the fact that TREX was identified and characterized about a decade ago, it had remained enigmatic how TREX is recruited to genes. The conserved Prp19 complex (Prp19C) has long been known for its function in splicing. We recently identified Prp19C to be essential for a second step in gene expression namely TREX occupancy at transcribed genes, answering this long-standing question but also raising new ones.

Different steps in gene expression are intimately linked. In Saccharomyces cerevisiae, the conserved TREX complex couples transcription to nuclear messenger RNA (mRNA) export. However, it is unknown how TREX is recruited to actively transcribed genes. Here, we show that the Prp19 splicing complex functions in transcription elongation. The Prp19 complex is recruited to transcribed genes, interacts with RNA polymerase II (RNAPII) and TREX, and is absolutely required for TREX occupancy at transcribed genes. Importantly, the Prp19 complex is necessary for full transcriptional activity. Taken together, we identify the Prp19 splicing complex as a novel transcription elongation factor that is essential for TREX occupancy at transcribed genes and that thus provides a novel link between transcription and messenger ribonucleoprotein (mRNP) formation.

The MR (Mre11 nuclease and Rad50 ABC ATPase) complex is an evolutionarily conserved sensor for DNA double-strand breaks, highly genotoxic lesions linked to cancer development. MR can recognize and process DNA ends even if they are blocked and misfolded. To reveal its mechanism, we determined the crystal structure of the catalytic head of Thermotoga maritima MR and analyzed ATP-dependent conformational changes. MR adopts an open form with a central Mre11 nuclease dimer and two peripheral Rad50 molecules, a form suited for sensing obstructed breaks. The Mre11 C-terminal helix-loop-helix domain binds Rad50 and attaches flexibly to the nuclease domain, enabling large conformational changes. ATP binding to the two Rad50 subunits induces a rotation of the Mre11 helix-loop-helix and Rad50 coiled-coil domains, creating a clamp conformation with increased DNA-binding activity. The results suggest that MR is an ATP-controlled transient molecular clamp at DNA double-strand breaks., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Multiple DNA-associated processes such as DNA repair, replication, and recombination are crucial for the maintenance of genome integrity. Here, we show a novel interaction between the transcription elongation factor Bur1-Bur2 and replication protein A (RPA), the eukaryotic single-stranded DNA-binding protein with functions in DNA repair, recombination, and replication. Bur1 interacted via its C-terminal domain with RPA, and bur1-ΔC mutants showed a deregulated DNA damage response accompanied by increased sensitivity to DNA damage and replication stress as well as increased levels of persisting Rad52 foci. Interestingly, the DNA damage sensitivity of an rfa1 mutant was suppressed by bur1 mutation, further underscoring a functional link between these two protein complexes. The transcription elongation factor Bur1-Bur2 interacts with RPA and maintains genome integrity during DNA replication stress.

Diverse steps in gene expression are tightly coupled. Curiously, the La-motif-containing protein Sro9 has been shown to play a role in transcription and translation. Here, we show that Sro9 interacts with nuclear and cytoplasmic protein complexes involved in gene expression. In addition, Sro9 shuttles between nucleus and cytoplasm and is exported from the nucleus in an mRNA export-dependent manner. Importantly, Sro9 is recruited to transcribed genes. However, whole genome expression analysis shows that loss of Sro9 function does not greatly change the level of specific transcripts indicating that Sro9 does not markedly affect their synthesis and/or stability. Taken together, Sro9 might bind to the mRNP already during transcription and accompany the mature mRNP to the cytoplasm where it modulates translation of the mRNA.

Yeast RNA polymerase (Pol) II consists of a 10-subunit core enzyme and the Rpb4/7 subcomplex, which is dispensable for catalytic activity and dissociates in vitro. To investigate whether Rpb4/7 is an integral part of DNA-associated Pol II in vivo, we used chromatin immunoprecipitation coupled to high resolution tiling microarray analysis. We show that the genome-wide occupancy profiles for Rpb7 and the core subunit Rpb3 are essentially identical. Thus, the complete Pol II associates with DNA in vivo, consistent with functional roles of Rpb4/7 throughout the transcription cycle.

A combination of crystallography, biochemistry, and gene expression analysis identifies the coactivator subcomplex Med8C/18/20 as a functionally distinct submodule of the Mediator head module. Med8C forms a conserved alpha-helix that tethers Med18/20 to the Mediator. Deletion of Med8C in vivo results in dissociation of Med18/20 from Mediator and in loss of transcription activity of extracts. Deletion of med8C, med18, or med20 causes similar changes in the yeast transcriptome, establishing Med8C/18/20 as a predominantly positive, gene-specific submodule required for low transcription levels of nonactivated genes, including conjugation genes. The presented structure-based system perturbation is superior to gene deletion analysis of gene regulation.

Translation is a highly complex process that is regulated by a multitude of factors. Here, we show that the conserved kinase Ctk1 functions in translation by enhancing decoding fidelity. Ctk1 associates with translating ribosomes in vivo and is needed for efficient translation. Ctk1 phosphorylates Rps2, a protein of the small ribosomal subunit, on Ser 238. Importantly, Ctk1-depleted as well as rps2-S238A mutant cells show a defect in translation elongation through an increase in the frequency of miscoding. The role of Ctk1 in translation may be conserved as the mammalian homolog of Ctk1, CDK9, also associates with polysomes. Since Ctk1 interacts with the TREX (transcription and mRNA export) complex, which couples transcription to mRNA export, Ctk1/CDK9 might bind to correctly processed mRNPs during transcription and accompany the mRNP to the ribosomes in the cytoplasm, where Ctk1 enhances efficient and accurate translation of the mRNA.

The bovine form of the autosomal recessive neurodegenerative disease spinal muscular atrophy (SMA) shows striking similarity to the human form of the disease. It has, however, been mapped to a genomic region not harboring the bovine orthologue of the SMN gene, mutation of which causes human SMA. After refinement of the mapping results we analyzed positional and functional candidate genes. One of three candidate genes, FVT1, encoding 3-ketodihydrosphingosine reductase, which catalyzes a crucial step in the glycosphingolipid metabolism, showed a G-to-A missense mutation that changes Ala-175 to Thr. The identified mutation is limited to SMA-affected animals and carriers and always appears in context of the founder haplotype. The Ala variant found in healthy animals showed the expected 3-ketodihydrosphingosine reductase activity in an in vitro enzyme assay. Importantly, the Thr variant found in SMA animals showed no detectable activity. Surprisingly, in an in vivo assay the mutated gene complements the growth defect of a homologous yeast knockout strain as well as the healthy variant. This finding explains the viability of affected newborn calves and the later neuron-specific onset of the disease, which might be due to the high sensitivity of these neurons to changes in housekeeping functions. Taken together, the described mutation in FVT1 is a strong candidate for causality of SMA in cattle. This result provides an animal model for understanding the underlying mechanisms of the development of SMA and will allow efficient selection against the disease in cattle.

The conserved TREX complex couples transcription to nuclear mRNA export. Here, we report that the uncharacterized open reading frame YOR166c genetically interacts with TREX complex components and encodes a novel protein named Swt1 for "synthetically lethal with TREX." Co-immunoprecipitation experiments show that Swt1 also interacts with the TREX complex biochemically. Consistent with a potential role in transcription as suggested by its interaction with TREX, Swt1 localizes mainly to the nucleus. Importantly, deletion of Swt1 leads to decreased transcription. Taken together, these data suggest that Swt1 functions in gene expression in conjunction with the TREX complex.

The Mediator head module stimulates basal RNA polymerase II (Pol II) transcription and enables transcriptional regulation. Here we show that the head subunits Med8, Med18 and Med20 form a subcomplex (Med8/18/20) with two submodules. The highly conserved N-terminal domain of Med8 forms one submodule that binds the TATA box-binding protein (TBP) in vitro and is essential in vivo. The second submodule consists of the C-terminal region of Med8 (Med8C), Med18 and Med20. X-ray analysis of this submodule reveals that Med18 and Med20 form related beta-barrel folds. A conserved putative protein-interaction face on the Med8C/18/20 submodule includes sites altered by srb mutations, which counteract defects resulting from Pol II truncation. Our results and published data support a positive role of the Med8/18/20 subcomplex in initiation-complex formation and suggest that the Mediator head contains a multipartite TBP-binding site that can be modulated by transcriptional activators.

The TREX (transcription/export) complex couples transcription elongation to the nuclear export of mRNAs. In this article, we show that the poly(A)(+) RNA-binding proteins Gbp2 and Hrb1, which resemble the serine-arginine-rich (SR) family of splicing factors found in higher eukaryotes, are specifically associated with the yeast TREX complex. We also show that Gbp2 and Hrb1 interact with Ctk1, a kinase that phosphorylates the C-terminal domain of RNA polymerase II during transcription elongation. Consistent with these findings, Gbp2 and Hrb1 associate with actively transcribed genes throughout their entire lengths. By using an RNA immunoprecipitation assay, we show that Gbp2 and Hrb1 also are bound to transcripts that are derived from these genes. We conclude that recruitment of the SR-like proteins Gbp2 and Hrb1 to mRNA occurs cotranscriptionally by means of association with the TREX complex and/or Ctk1.

Yra1p and Sub2p are components of the TREX complex, which couples transcription elongation with nuclear export of mRNAs. Here, we report a genetic interaction between Yra1p and a conserved protein Sac3p, which previously was found to interact with Sub2p. In vivo, Sac3p forms a stable complex with Thp1p, which was reported to function in transcription elongation. In addition, Sac3p binds to the mRNA exporter Mex67p-Mtr2p and requires the nucleoporin Nup1p to dock at the nuclear side of the nuclear pore complex (NPC). Significantly, mutations in Sac3p or Thp1p lead to strong mRNA export defects. Taken together, our data suggest that the novel Sac3p-Thp1p complex functions by docking the mRNP to specific nucleoporins at the nuclear entrance of the NPC.

The essential yeast proteins Yra1 and Sub2 are messenger RNA export factors that have conserved counterparts in metazoans, designated Aly and UAP56, respectively. These factors couple the machineries that function in splicing and export of mRNA. Here we show that both Yra1 and Sub2 are stoichiometrically associated with the heterotetrameric THO complex, which functions in transcription in yeast. We also show that Sub2 and Yra1 interact genetically with all four components of the THO complex (Tho2, Hpr1, Mft1 and Thp2). Moreover, these components operate in the export of bulk poly(A)(+) RNA as well as of mRNA derived from intronless genes. Both Aly and UAP56 associate with human counterparts of the THO complex. Together, these data define a conserved complex, designated the TREX ('transcription/export') complex. The TREX complex is specifically recruited to activated genes during transcription and travels the entire length of the gene with RNA polymerase II. Our data indicate that the TREX complex has a conserved role in coupling transcription to mRNA export.

Yra1p is an essential and conserved mRNA export factor in yeast. Strikingly, removal of the intron from YRA1 causes a dominant-negative growth phenotype and a concomitant inhibition of mRNA export. However, both defects are neutralized by replacement of the intron of YRA1 by a different intron. Significantly, Yra1p is overproduced in yeast when expressed from its intronless gene, but Yra1p levels are the same as the wild type when expressed from an intron-containing YRA1 gene. Thus, an intron in YRA1 controls Yra1p expression and mRNA export.

The yeast nuclear protein Yra1p is an essential export factor for mRNA. Yra1p interacts directly with the mRNA transport factor Mex67p/Mtr2p, which is associated with the nuclear pore. Here, we report a genetic interaction between YRA1 and SUB2, the gene for a DEAD box helicase involved in splicing. Mutation of SUB2 as well as its overexpression leads to a defect in mRNA export. Moreover, Yra1p and Sub2p bind directly to each other both in vivo and in vitro. Significantly, Sub2p and Mex67p/Mtr2p bind to the same domains of Yra1p, and the proteins compete for binding to Yra1p. Together, these data indicate that the spliceosomal component Sub2p is also important in mRNA export and may function to recruit Yra1p to the mRNA. Sub2p may then be displaced from Yra1p by the binding of Mex67p/Mtr2p, which participates in the export of mRNA through the nuclear pores.

It is not known how Mex67p and Mtr2p, which form a heterodimer essential for mRNA export, transport mRNPs through the nuclear pore. Here, we show that the Mex67p/Mtr2p complex binds to all of the repeat types (GLFG, FXFG, and FG) found in nucleoporins. For this interaction, complex formation between Mex67p and Mtr2p has to occur. MEX67 and MTR2 also genetically interact with different types of repeat nucleoporins, such as Nup116p, Nup159p, Nsp1p, and Rip1p/Nup40p. These data suggest a model in which nuclear mRNA export requires the Mex67p/Mtr2p heterodimeric complex to directly contact several repeat nucleoporins, organized in different nuclear pore complex subcomplexes, as it carries the mRNP cargo through the nuclear pore.

Mex67p and Mtr2p constitute an essential mRNA export complex that interacts with poly(A)+ RNA and nuclear pore proteins. We have identified Yra1p, an intranuclear protein with in vitro RNA-RNA annealing activity, which directly binds to Mex67p. The complex between Yra1p and Mex67p was reconstituted in vitro and shown by UV-crosslinking to bind directly to RNA. Mutants of YRA1 are impaired in nuclear poly(A)+ RNA export at restrictive growth conditions. ALY, the mouse homologue of Yra1p and a transcriptional coactivator, can bind in vitro to yeast and human Mex67p and partly complements the otherwise non-viable yra1 null mutant. Thus, Yra1p is the first RNA-binding protein characterized, which bridges the shuttling Mex67p/Mtr2p exporter to intranuclear mRNA transport cargoes.

Eukaryotic cells massively exchange macromolecules (proteins and RNAs) between the nucleus and cytoplasm through the nuclear pore complexes. Whereas a mechanistic picture emerges of how proteins are imported into and exported from the nucleus, less is known about nuclear exit of the different classes of RNAs. However, the yeast Saccharomyces cerevisiae offers an experimental system to study nuclear RNA export in vivo and thus to genetically dissect the different RNA export machineries. In this review, we summarize our current knowledge and recent progress in identifying components involved in nuclear RNA export in yeast.

Human TAP is an orthologue of the yeast mRNA export factor Mex67p. In mammalian cells, TAP has a preferential intranuclear localization, but can also be detected at the nuclear pores and shuttles between the nucleus and the cytoplasm. TAP directly associates with mRNA in vivo, as it can be UV-crosslinked to poly(A)+ RNA in HeLa cells. Both the FG-repeat domain of nucleoporin CAN/Nup214 and a novel human 15 kDa protein (p15) with homology to NTF2 (a nuclear transport factor which associates with RanGDP), directly bind to TAP. When green fluorescent protein (GFP)-tagged TAP and p15 are expressed in yeast, they localize to the nuclear pores. Strikingly, co-expression of human TAP and p15 restores growth of the otherwise lethal mex67::HIS3/mtr2::HIS3 double knockout strain. Thus, the human TAP-p15 complex can functionally replace the Mex67p-Mtr2p complex in yeast and thus performs a conserved role in nuclear mRNA export.