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

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

An introduction to video methods in organizational research

Video has become a methodological tool of choice for many researchers in social science, but video methods are relatively new to the field of organization studies. This article is an introduction to video methods. First, we situate video methods relative to other kinds of research, suggesting that video recordings and analyses can be used to replace or supplement other approaches, not only observational studies but also retrospective methods such as interviews and surveys. Second, we describe and discuss various features of video data in relation to ontological assumptions that researchers may bring to their research design. Video involves both opportunities and pitfalls for researchers, who ought to use video methods in ways that are consistent with their assumptions about the world and human activity. Third, we take a critical look at video methods by reporting progress that has been made while acknowledging gaps and work that remains to be done. Our critical considerations point repeatedly at articles in this special issue, which represent recent and important advances in video method

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

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

Npl3 physically interacts with Bre1.

<p>Co-immunoprecipitation analyses of Npl3 and members of the histone H2B ubiquitination machinery. Whole cell extracts from strains with the indicated proteins endogenously tagged with HA or GFP were immunoprecipitated with an α-Npl3 antibody <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003101#pgen.1003101-Siebel1" target="_blank">[63]</a> or non-specific antibody (α–n.s.). Western blot using α-HA or α-GFP from each co-IP experiment is shown. The sensitivity of the interaction to RNase (lane 4, +RNaseA) was determined by treating lysates with RNase A prior to immunoprecipitation. Lane 1 shows 1/60 total sample for each lysate. Bottom panel confirms presence of Npl3 in the immunoprecipitate.</p

A chromatin-centered survey of Npl3 genetic interactions.

<p>Summary of chromatin and transcription factors that exhibit genetic interactions with a deletion of <i>NPL3</i>. Colored ovals represent subunits identified in the SGA screens or by directed genetics; red indicates a suppressive (positive) interaction and green indicates a synthetic (negative) interaction. Outlined ovals refer to the complex that individual subunits belong to. K123Ub refers to the <i>htb1K123R</i> point mutant. K36me refers the <i>hht1K36A</i> point mutant. Grey or white indicates the genetic interaction was not tested. Physical interactions tested by co-IP are Npl3:Bre1 (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003101#pgen-1003101-g003" target="_blank">Figure 3</a>) and Npl3:U1 <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003101#pgen.1003101-Kress1" target="_blank">[7]</a>. Bold rectangle indicates factors shown in this paper and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003101#pgen.1003101-Kress1" target="_blank">[7]</a> to promote splicing; whether the presence of Npl3 can influence local H2B ubiquitination levels or dynamics remains unresolved. The PolII C-terminal domain is drawn in grey.</p

Genetic interactions between H2B ubiquitination machinery and canonical splicing factors.

<p>(A) Synthetic growth analyses between <i>bre1Δ</i> and genes encoding splicing factors. Double mutants were generated by tetrad dissection; log-phase cultures of the indicated strains were serially diluted and grown at the indicated temperatures. To the right of panels is the name of the spliceosomal complex to which the splicing factor mutants belong. NTC: Nineteen Complex. (B) Growth analyses of <i>ubp8Δ</i> and genes encoding splicing factors. Double mutants were generated and analyzed as in (A).</p