CWC22 Supports Pre-mRNA Splicing and Exon Junction Complex Assembly

The exon junction complex (EJC) is a multi-protein complex that assembles on mRNA during splicing. It binds upstream of exon-exon junctions and preserves the positional information of the former splice sites within the mRNP architecture. In metazoan cells, the EJC coordinates several key aspects of gene expression, such as mRNA export, cytoplasmic localization, translation efficiency and quality control by nonsense-mediated mRNA decay (NMD). Although the composition of EJCs is well understood, the mechanism mediating splicing-dependent EJC assembly and the factor(s) recruiting the EJC to the spliceosome remain elusive. Here, I describe the identification of CWC22 as an essential splicing factor that is required for EJC assembly in human cells. The central domain (MIF4G) of CWC22 directly interacts with the EJC core protein eIF4A3, and this interaction is reminiscent of the eIF4A/eIF4G translation initiation complex. The interaction with CWC22 recruits eIF4A3 to the early spliceosomal complex and initiates EJC assembly. Mutations in eIF4A3 that disrupt the interaction with CWC22 also prevent splicing-dependent EJC assembly, but do not affect the formation of splicing-independent EJCs from recombinant components in vitro. Interestingly, CWC22 is also required for pre-mRNA splicing, and unspliced pre-mRNAs accumulate in CWC22-depleted cells. Complementation assays revealed that a central segment of CWC22, comprising of the MIF4G and an adjacent MA3 domain, supports pre-mRNA splicing in CWC22-depleted cells. By regulating both pre-mRNA splicing and EJC assembly, CWC22 plays a dual role in mammalian gene expression. The two functions of CWC22 can be experimentally separated, since over-expression of a CWC22-mutant that is unable to bind eIF4A3 inhibits EJC assembly without affecting pre-mRNA splicing. Over-expression of this mutant is highly toxic to cells, demonstrating the importance of EJCs for gene regulation in human cells. Taken together, CWC22 represents the first direct link between the splicing machinery and the EJC, and the discovery of its dual function during pre-mRNA splicing highlights the complex interconnection of individual processing steps during mammalian gene expression

Studying the composition of mRNPs in vitro using splicing-competent cell extracts

The correct processing and faithful decoding of mRNAs during gene expression depends on the interaction with RNA-binding proteins (RBPs). The association of RBPs with pre-mRNAs starts during transcription by RNA polymerase II and undergoes constant remodeling during pre-mRNA processing and later steps of genes expression. Recently developed high throughput methods enabled to define RBP binding sites in vivo and to identify a large number of novel RBPs in eukaryotic cells. However, the detailed characterization of RBP-RNA interactions as well as the analysis of functional RNPs is greatly facilitated by well-defined in vitro systems. Here, we describe a versatile method to study the assembly and splicing-dependent remodeling of mRNPs in vitro. This method employs splicing-competent whole cell extracts (WCE) generated from transfected human embryonic kidney (HEK) 293 cells. FLAG-tagged proteins present in the WCE are incorporated into mRNPs in vitro and afterwards used to immunoprecipitate substrate RNAs. We outline the principles of purifying in vitro assembled mRNPs and provide detailed protocols for the preparation and use of whole cell extracts. Alternative purification strategies and RNA substrates are discussed. (C) 2013 Elsevier Inc. All rights reserved

Exoribonuclease-Resistant RNAs Exist within both Coding and Noncoding Subgenomic RNAs

During infection, viruses often produce subgenomic RNAs (sgRNAs) that either serve as the template for protein synthesis or act as “riboregulators” that interact with and influence the viral and cellular machinery. Recently, a mechanism for producing sgRNAs was found that depends on the presence of specifically structured RNA elements (xrRNAs). However, the degree to which this mechanism is used, where the elements are found, their structural diversity, and what types of sgRNAs are produced by this pathway were unclear. This article describes the discovery of these structured RNA elements in two large families of plant viruses and shows that they are used to produce both protein-coding sgRNAs and “riboregulatory” RNAs. These discoveries provide evidence that xrRNA-based RNA maturation pathways may be more widespread than previously anticipated and that they are involved in producing a variety of RNAs of diverse functions.Many viruses produce protein-coding and noncoding subgenomic RNAs (sgRNAs) that are critical for infection. A recently discovered pathway for viral sgRNA production uses exoribonuclease-resistant RNAs (xrRNAs), discrete folded RNA elements that block the processive exoribonucleolytic degradation of RNA. xrRNAs are widespread in animal-infecting flaviviruses but had been found only in three members of the plant virus genus Dianthovirus. Also, xrRNAs had been found only in the 3′ untranslated regions (3′UTRs) of viral RNAs, where they produce noncoding sgRNAs. The degree to which xrRNA elements exist in other viruses, the conservation of their ring-like fold, and the ability of xrRNAs to operate in diverse contexts were unknown. Using computational tools and biochemical assays, we discovered xrRNA elements pervading two large families of plant-infecting RNA viruses, demonstrating their importance and widespread utility. Comparison of the sequences and functional requirements suggests that all adopt the characteristic ring-like fold. Unexpectedly, many of these newly discovered xrRNAs are located in intergenic regions rather than 3´UTRs, and some are associated with the 5′ ends of subgenomic RNAs that encode viral proteins. This suggests that xrRNAs are involved in the production of both coding and noncoding subgenomic RNAs and can operate as part of broader mechanisms to regulate RNA levels and protein expression. These discoveries expand the potential roles for xrRNAs and suggest that xrRNAs may represent a more general strategy for RNA maturation and maintenance than previously known

CWC22 Connects Pre-mRNA Splicing and Exon Junction Complex Assembly

The exon junction complex (EJC) is a key regulator of posttranscriptional mRNA fate and binds to mRNA during splicing. Although the composition of EJCs is well understood, the mechanism mediating splicing-dependent EJC assembly and the factor(s) recruiting the EJC remain elusive. Here, we identify CWC22 as an essential splicing factor that is required for EJC assembly. In CWC22-depleted cells, pre-mRNA splicing is impaired but is rescued by a central fragment of CWC22. We show that the MIF4G domain of CWC22 initiates EJC assembly via a direct interaction with the EJC core protein eIF4A3, and we characterize mutations in eIF4A3 that abolish binding to CWC22. These eIF4A3 mutants efficiently nucleate splicing-independent recombinant EJC core complexes, but they fail to support splicing-dependent EJC deposition. Our work establishes a direct link between the splicing machinery and the EJC, hence uncovering a molecular interaction at the center of a posttranscriptional gene regulation network

A short conserved motif in ALYREF directs cap- and EJC-dependent assembly of export complexes on spliced mRNAs

The export of messenger RNAs (mRNAs) is the final of several nuclear posttranscriptional steps of gene expression. The formation of export-competent mRNPs involves the recruitment of export factors that are assumed to facilitate transport of the mature mRNAs. Using in vitro splicing assays, we show that a core set of export factors, including ALYREF, UAP56 and DDX39, readily associate with the spliced RNAs in an EJC (exon junction complex)- and cap-dependent manner. In order to elucidate how ALYREF and other export adaptors mediate mRNA export, we conducted a computational analysis and discovered four short, conserved, linear motifs present in RNA-binding proteins. We show that mutation in one of the new motifs (WxHD) in an unstructured region of ALYREF reduced RNA binding and abolished the interaction with eIF4A3 and CBP80. Additionally, the mutation impaired proper localization to nuclear speckles and export of a spliced reporter mRNA. Our results reveal important details of the orchestrated recruitment of export factors during the formation of export competent mRNPs

The crystal structure of a Polerovirus exoribonuclease-resistant RNA shows how diverse sequences are integrated into a conserved fold.

Exoribonuclease-resistant RNAs (xrRNAs) are discrete elements that block the progression of 5' to 3' exoribonucleases using specifically folded RNA structures. A recently discovered class of xrRNA is widespread in several genera of plant-infecting viruses, within both noncoding and protein-coding subgenomic RNAs. The structure of one such xrRNA from a dianthovirus revealed three-dimensional details of the resistant fold but did not answer all questions regarding the conservation and diversity of this xrRNA class. Here, we present the crystal structure of a representative polerovirus xrRNA that contains sequence elements that diverge from the previously solved structure. This new structure rationalizes previously unexplained sequence conservation patterns and shows interactions not present in the first structure. Together, the structures of these xrRNAs from dianthovirus and polerovirus genera support the idea that these plant virus xrRNAs fold through a defined pathway that includes a programmed intermediate conformation. This work deepens our knowledge of the structure-function relationship of xrRNAs and shows how evolution can craft similar RNA folds from divergent sequences

A short conserved motif in ALYREF directs cap- and EJC-dependent assembly of export complexes on spliced mRNAs

The export of messenger RNAs (mRNAs) is the final of several nuclear posttranscriptional steps of gene expression. The formation of export-competent mRNPs involves the recruitment of export factors that are assumed to facilitate transport of the mature mRNAs. Using in vitro splicing assays, we show that a core set of export factors, including ALYREF, UAP56 and DDX39, readily associate with the spliced RNAs in an EJC (exon junction complex)- and cap-dependent manner. In order to elucidate how ALYREF and other export adaptors mediate mRNA export, we conducted a computational analysis and discovered four short, conserved, linear motifs present in RNA-binding proteins. We show that mutation in one of the new motifs (WxHD) in an unstructured region of ALYREF reduced RNA binding and abolished the interaction with eIF4A3 and CBP80. Additionally, the mutation impaired proper localization to nuclear speckles and export of a spliced reporter mRNA. Our results reveal important details of the orchestrated recruitment of export factors during the formation of export competent mRNPs

NKAP is a novel RS-related protein that interacts with RNA and RNA binding proteins

NKAP is a highly conserved protein with roles in transcriptional repression, T-cell development, maturation and acquisition of functional competency and maintenance and survival of adult hematopoietic stem cells. Here we report the novel role of NKAP in splicing. With NKAP-specific antibodies we found that NKAP localizes to nuclear speckles. NKAP has an RS motif at the N-terminus followed by a highly basic domain and a DUF 926 domain at the C-terminal region. Deletion analysis showed that the basic domain is important for speckle localization. In pull-down experiments, we identified RNA-binding proteins, RNA helicases and splicing factors as interaction partners of NKAP, among them FUS/TLS. The FUS/TLS-NKAP interaction takes place through the RS domain of NKAP and the RGG1 and RGG3 domains of FUS/TLS. We analyzed the ability of NKAP to interact with RNA using in vitro splicing assays and found that NKAP bound both spliced messenger RNA (mRNA) and unspliced pre-mRNA. Genome-wide analysis using crosslinking and immunoprecipitation-seq revealed NKAP association with U1, U4 and U5 small nuclear RNA, and we also demonstrated that knockdown of NKAP led to an increase in pre-mRNA percentage. Our results reveal NKAP as nuclear speckle protein with roles in RNA splicing and processing