Summary 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.

Cytoplasmic localization of mRNA molecules is a powerful mechanism for generating cell polarity. In vertebrates, one paradigm is localization of Vg1 RNA within the Xenopus oocyte, a process directed by recognition of a localization element within the Vg1 3' UTR. We show that specific base changes within the localization element abolish both localization in vivo and binding in vitro by a single protein, VgRBP60. VgRBP60 is homologous to a human hnRNP protein, hnRNP I, and combined immunolocalization and in situ hybridization demonstrate striking colocalization of hnRNP I and Vg1 RNA within the vegetal cytoplasm of the Xenopus oocyte. These results implicate a novel role in cytoplasmic RNA transport for this family of nuclear RNA-binding proteins.

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.

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 nucleocytoplasmic 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. T he heterogeneous nuclear ribonucleoprotein particle (hnRNP) proteins are involved in a variety of processes in mRNA metabolism including pre-mRNA splicing, mRNA transport, and translation (1). These processes are often regulated by cellular signaling pathways, but how this dynamic control is achieved is mostly unknown. Some hnRNP proteins are known to be phosphorylated, but in most cases the particular kinase that modifies the protein is not known. Moreover, it is generally not clear how such modifications affect protein function. HnRNP proteins localize primarily in the nucleus at steady state but some of them engage in nucleo-cytoplasmic shuttling (2, 3). There are examples of hnRNP localization being altered by specific signaling pathways (4, 5).The polypyrimidine tract-binding protein (PTB, or hnRNP I) has both nuclear and cytoplasmic functions. In the nucleus, it is a splicing repressor of a number of alternative exons (6, 7). In the cytoplasm, PTB plays a role in viral RNA translation through internal ribosome entry sites (8-11). Also in the cytoplasm, PTB is implicated in mRNA localization in Xenopus oocytes (12). The protein contains four RNA-recognition-motif type RNA binding domains (RRMs) and a conserved N-terminal domain. The N-terminal 55-aa segment of PTB contains both nuclear import and export signals and is sufficient to allow some nucleocytoplasmic shuttling in heterokaryon assays (13-17). There is an additional sequence within RRM2 that enhances nuclear export (17). At steady state, PTB is highly enriched in the nucleus, but its distribution must be regulated because the protein also has cytoplasmic functions. However, little is known about this regulation and what cellular signaling...

Summary 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 co-transcriptional recruitment of splicing factors. Using chromatin immunoprecipitation, we showed that mutation of NPL3 reduces the occupancy of U1 and U2 snRNPs at at genes whose splicing is stimulated by Npl3. This result provides strong evidence that an SR protein can promote recruitment of splicing factors to chromatin.

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.

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.

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.