The histone variant H2A.Z promotes efficient cotranscriptional splicing in S. cerevisiae.

In eukaryotes, a dynamic ribonucleic protein machine known as the spliceosome catalyzes the removal of introns from premessenger RNA (pre-mRNA). Recent studies show the processes of RNA synthesis and RNA processing to be spatio-temporally coordinated, indicating that RNA splicing takes place in the context of chromatin. H2A.Z is a highly conserved histone variant of the canonical histone H2A. In Saccharomyces cerevisiae, H2A.Z is deposited into chromatin by the SWR-C complex, is found near the 5' ends of protein-coding genes, and has been implicated in transcription regulation. Here we show that splicing of intron-containing genes in cells lacking H2A.Z is impaired, particularly under suboptimal splicing conditions. Cells lacking H2A.Z are especially dependent on a functional U2 snRNP (small nuclear RNA [snRNA] plus associated proteins), as H2A.Z shows extensive genetic interactions with U2 snRNP-associated proteins, and RNA sequencing (RNA-seq) reveals that introns with nonconsensus branch points are particularly sensitive to H2A.Z loss. Consistently, H2A.Z promotes efficient spliceosomal rearrangements involving the U2 snRNP, as H2A.Z loss results in persistent U2 snRNP association and decreased recruitment of downstream snRNPs to nascent RNA. H2A.Z impairs transcription elongation, suggesting that spliceosome rearrangements are tied to H2A.Z's role in elongation. Depletion of disassembly factor Prp43 suppresses H2A.Z-mediated splice defects, indicating that, in the absence of H2A.Z, stalled spliceosomes are disassembled, and unspliced RNAs are released. Together, these data demonstrate that H2A.Z is required for efficient pre-mRNA splicing and indicate a role for H2A.Z in coordinating the kinetics of transcription elongation and splicing.

Histone H3K36 methylation regulates pre-mRNA splicing in Saccharomyces cerevisiae.

Co-transcriptional splicing takes place in the context of a highly dynamic chromatin architecture, yet the role of chromatin restructuring in coordinating transcription with RNA splicing has not been fully resolved. To further define the contribution of histone modifications to pre-mRNA splicing in Saccharomyces cerevisiae, we probed a library of histone point mutants using a reporter to monitor pre-mRNA splicing. We found that mutation of H3 lysine 36 (H3K36) - a residue methylated by Set2 during transcription elongation - exhibited phenotypes similar to those of pre-mRNA splicing mutants. We identified genetic interactions between genes encoding RNA splicing factors and genes encoding the H3K36 methyltransferase Set2 and the demethylase Jhd1 as well as point mutations of H3K36 that block methylation. Consistent with the genetic interactions, deletion of SET2, mutations modifying the catalytic activity of Set2 or H3K36 point mutations significantly altered expression of our reporter and reduced splicing of endogenous introns. These effects were dependent on the association of Set2 with RNA polymerase II and H3K36 dimethylation. Additionally, we found that deletion of SET2 reduces the association of the U2 and U5 snRNPs with chromatin. Thus, our study provides the first evidence that H3K36 methylation plays a role in co-transcriptional RNA splicing in yeast.

A single SR-like protein, Npl3, promotes pre-mRNA splicing in budding yeast.

Mammalian SR proteins are a family of reversibly phosphorylated RNA binding proteins primarily studied for their roles in alternative splicing. While budding yeast lack alternative splicing, they do have three SR-like proteins: Npl3, Gbp2, and Hrb1. However, these have been best characterized for their roles in mRNA export, leaving their potential roles in splicing largely unexplored. Here, we combined high-density genetic interaction profiling and genome-wide splicing-sensitive microarray analysis to demonstrate that a single SR-like protein, Npl3, is required for efficient splicing of a large set of pre-mRNAs in Saccharomyces cerevisiae. We tested the hypothesis that Npl3 promotes splicing by facilitating cotranscriptional recruitment of splicing factors. Using chromatin immunoprecipitation, we showed that mutation of NPL3 reduces the occupancy of U1 and U2 snRNPs at genes whose splicing is stimulated by Nbl3. This result provides strong evidence that an SR protein can promote recruitment of splicing factors to chromatin.

A genetic interaction map of RNA-processing factors reveals links between Sem1/Dss1-containing complexes and mRNA export and splicing.

We used a quantitative, high-density genetic interaction map, or E-MAP (Epistatic MiniArray Profile), to interrogate the relationships within and between RNA-processing pathways. Due to their complexity and the essential roles of many of the components, these pathways have been difficult to functionally dissect. Here, we report the results for 107,155 individual interactions involving 552 mutations, 166 of which are hypomorphic alleles of essential genes. Our data enabled the discovery of links between components of the mRNA export and splicing machineries and Sem1/Dss1, a component of the 19S proteasome. In particular, we demonstrate that Sem1 has a proteasome-independent role in mRNA export as a functional component of the Sac3-Thp1 complex. Sem1 also interacts with Csn12, a component of the COP9 signalosome. Finally, we show that Csn12 plays a role in pre-mRNA splicing, which is independent of other signalosome components. Thus, Sem1 is involved in three separate and functionally distinct complexes.

The yeast SR-like protein Npl3 links chromatin modification to mRNA processing.

Eukaryotic gene expression involves tight coordination between transcription and pre-mRNA splicing; however, factors responsible for this coordination remain incompletely defined. Here, we explored the genetic, functional, and biochemical interactions of a likely coordinator, Npl3, an SR-like protein in Saccharomyces cerevisiae that we recently showed is required for efficient co-transcriptional recruitment of the splicing machinery. We surveyed the NPL3 genetic interaction space and observed a significant enrichment for genes involved in histone modification and chromatin remodeling. Specifically, we found that Npl3 genetically interacts with both Bre1, which mono-ubiquitinates histone H2B as part of the RAD6 Complex, and Ubp8, the de-ubiquitinase of the SAGA Complex. In support of these genetic data, we show that Bre1 physically interacts with Npl3 in an RNA-independent manner. Furthermore, using a genome-wide splicing microarray, we found that the known splicing defect of a strain lacking Npl3 is exacerbated by deletion of BRE1 or UBP8, a phenomenon phenocopied by a point mutation in H2B that abrogates ubiquitination. Intriguingly, even in the presence of wild-type NPL3, deletion of BRE1 exhibits a mild splicing defect and elicits a growth defect in combination with deletions of early and late splicing factors. Taken together, our data reveal a connection between Npl3 and an extensive array of chromatin factors and describe an unanticipated functional link between histone H2B ubiquitination and pre-mRNA splicing.

Nuclear RNP complex assembly initiates cytoplasmic RNA localization.

Cytoplasmic localization of mRNAs is a widespread mechanism for generating cell polarity and can provide the basis for patterning during embryonic development. A prominent example of this is localization of maternal mRNAs in Xenopus oocytes, a process requiring recognition of essential RNA sequences by protein components of the localization machinery. However, it is not yet clear how and when such protein factors associate with localized RNAs to carry out RNA transport. To trace the RNA-protein interactions that mediate RNA localization, we analyzed RNP complexes from the nucleus and cytoplasm. We find that an early step in the localization pathway is recognition of localized RNAs by specific RNA-binding proteins in the nucleus. After transport into the cytoplasm, the RNP complex is remodeled and additional transport factors are recruited. These results suggest that cytoplasmic RNA localization initiates in the nucleus and that binding of specific RNA-binding proteins in the nucleus may act to target RNAs to their appropriate destinations in the cytoplasm.

Conserved and clustered RNA recognition sequences are a critical feature of signals directing RNA localization in Xenopus oocytes.

Although it is widely regarded that the targeting of RNA molecules to subcellular destinations depends upon the recognition of cis-elements found within their 3' untranslated regions (UTR), relatively little is known about the specific features of these cis-sequences that underlie their function. Interaction between specific repeated motifs within the 3' UTR and RNA-binding proteins has been proposed as a critical step in the localization of Vg1 RNA to the vegetal pole of Xenopus oocytes. To understand the relative contributions of repeated localization element (LE) sequences, we used comparative functional analysis of Vg1 LEs from two frog species, Xenopus laevis and Xenopus borealis. We show that clusters of repeated VM1 and E2 motifs are required for efficient localization. However, groups of either site alone are not sufficient for localization. In addition, we present evidence that the X. borealis Vg1 LE is recognized by the same set of RNA-binding proteins as the X. laevis Vg1 LE and is capable of productive interactions with the X. laevis transport machinery as it is sufficient to direct vegetal localization in X. laevis oocytes. These results suggest that clustered sets of cis-acting sites within the LE direct vegetal transport through specific interactions with the localization machinery.

Autoregulation of Npl3, a yeast SR protein, requires a novel downstream region and serine phosphorylation.

Npl3 is an SR-like protein with documented roles in mRNA export and transcription termination. Maintaining appropriate levels of Npl3 protein is critical for cell survival. Here we show that Npl3 negatively regulates its own expression via modulation of its mRNA levels. By creating gene chimeras, we demonstrate that the region downstream of the coding sequence of Npl3 is necessary and sufficient to confer regulation. The use of different polyadenylation sites in this region results in at least two stable RNAs; read-through of these sites causes the formation of 3'-extended RNAs that are highly unstable and therefore largely unproductive. Increasing the amount of Npl3 protein promotes read-through. Notably, the loss of Npl3 phosphorylation promotes the use of the productive polyadenylation sites, resulting in elevated levels of Npl3 protein. We propose that proper levels of Npl3 protein are achieved by a negative feedback loop in which phosphorylated Npl3 suppresses efficient recognition of the productive processing signals in its own transcript.

Protein kinase A phosphorylation modulates transport of the polypyrimidine tract-binding protein.

The heterogeneous nuclear ribonucleoprotein particle (hnRNP) proteins play important roles in mRNA processing in eukaryotes, but little is known about how they are regulated by cellular signaling pathways. The polypyrimidine-tract binding protein (PTB, or hnRNP I) is an important regulator of alternative pre-mRNA splicing, of viral RNA translation, and of mRNA localization. Here we show that the nucleo-cytoplasmic transport of PTB is regulated by the 3',5'-cAMP-dependent protein kinase (PKA). PKA directly phosphorylates PTB on conserved Ser-16, and PKA activation in PC12 cells induces Ser-16 phosphorylation. PTB carrying a Ser-16 to alanine mutation accumulates normally in the nucleus. However, export of this mutant protein from the nucleus is greatly reduced in heterokaryon shuttling assays. Conversely, hyperphosphorylation of PTB by coexpression with the catalytic subunit of PKA results in the accumulation of PTB in the cytoplasm. This accumulation is again specifically blocked by the S16A mutation. Similarly, in Xenopus oocytes, the phospho-Ser-16-PTB is restricted to the cytoplasm, whereas the non-Ser-16-phosphorylated PTB is nuclear. Thus, direct PKA phosphorylation of PTB at Ser-16 modulates the nucleo-cytoplasmic distribution of PTB. This phosphorylation likely plays a role in the cytoplasmic function of PTB.

A consensus RNA signal that directs germ layer determinants to the vegetal cortex of Xenopus oocytes.

RNA localization is an important mechanism for generating cellular diversity and polarity in the early embryo. In Xenopus, the correct localization of the RNA encoding the T-box transcription factor VegT is essential for the correct spatial organization and identity of endoderm and mesoderm. Although localization signals in the 3' UTR have been identified for many localized RNAs, insight into what constitutes an RNA localization signal remains elusive. To investigate possible common features between signals that direct different RNAs to the same subcellular region, we carried out a detailed analysis of the uncharacterized VegT RNA localization signal and compared it with the well-studied Vg1 localization signal. Both RNAs localize to the vegetal cortex during the same period of oogenesis. Our results suggest a common RNA localization signal at the level of clustered redundant protein-binding motifs and trans-acting factors. We propose that what characterizes RNA localization signals in general is not the nucleotide sequence or secondary structure per se, but the critical clustering of specific redundant protein-binding motifs.