Cytoplasmic sorting of mRNAs by microtubule-based transport is widespread, yet very little is known at the molecular level about how specific transcripts are linked to motor complexes. In Drosophila, minus-end-directed transport of developmentally important transcripts by the dynein motor is mediated by seemingly divergent mRNA elements. Here we provide evidence that direct recognition of these mRNA localization signals is mediated by the Egalitarian (Egl) protein. Egl and the dynein cofactor Bicaudal-D (BicD) are the only proteins from embryonic extracts that are abundantly and specifically enriched on RNA localization signals from transcripts of gurken, hairy, K10, and the I factor retrotransposon. In vitro assays show that, despite lacking a canonical RNAbinding motif, Egl directly recognizes active localization elements. We also reveal a physical interaction between Egl and a conserved domain for cargo recruitment in BicD and present data suggesting that Egl participates selectively in BicD-mediated transport of mRNA in vivo. Our work leads to the first working model for a complete connection between minus-end-directed mRNA localization signals and microtubules and reveals molecular strategies that are likely to be of general relevance for cargo transport by dynein.[Keywords: Drosophila; dynein; mRNA localization; microtubule-based transport; stem-loop] Supplemental material is available at http://www.genesdev.org.

SummaryThe mechanisms by which the major Polycomb group (PcG) complexes PRC1 and PRC2 are recruited to target sites in vertebrate cells are not well understood. Building on recent studies that determined a reciprocal relationship between DNA methylation and Polycomb activity, we demonstrate that, in methylation-deficient embryonic stem cells (ESCs), CpG density combined with antagonistic effects of H3K9me3 and H3K36me3 redirects PcG complexes to pericentric heterochromatin and gene-rich domains. Surprisingly, we find that PRC1-linked H2A monoubiquitylation is sufficient to recruit PRC2 to chromatin in vivo, suggesting a mechanism through which recognition of unmethylated CpG determines the localization of both PRC1 and PRC2 at canonical and atypical target sites. We discuss our data in light of emerging evidence suggesting that PcG recruitment is a default state at licensed chromatin sites, mediated by interplay between CpG hypomethylation and counteracting H3 tail modifications.

Transcription through early-elongation checkpoints requires phosphorylation of negative transcription elongation factors (NTEFs) by the cyclin-dependent kinase (CDK)9. Using CDK9 inhibitors and global run-on sequencing (GRO-seq), we have mapped CDK9 inhibitor-sensitive checkpoints genome-wide in human (Homo sapiens) cells. Our data indicate that early-elongation checkpoints are a general feature of RNA polymerase (pol) II-transcribed human genes and occur independently of polymerase stalling. Pol II that has negotiated the early-elongation checkpoint can elongate in the presence of inhibitors but, remarkably, terminates transcription prematurely close to the terminal polyadenylation (poly(A)) site. Our analysis has revealed a hitherto-unsuspected poly(A)-associated elongation checkpoint, which has major implications for the regulation of gene expression. Interestingly, the pattern of modification of the carboxyl-terminal domain (CTD) of pol II terminated at this novel checkpoint largely mirrors the pattern normally found downstream of the poly(A) site, suggesting common mechanisms of termination.

No abstract

Human U1 small nuclear (sn)RNA, required for splicing of pre-mRNA, is encoded by genes on chromosome 1 (1p36). Imperfect copies of these U1 snRNA genes, also located on chromosome 1 (1q12-21), were thought to be pseudogenes. However, many of these “variant” (v)U1 snRNA genes produce fully processed transcripts. Using antisense oligonucleotides to block the activity of a specific vU1 snRNA in HeLa cells, we have identified global transcriptome changes following interrogation of the Affymetrix Human Exon ST 1.0 array. Our results indicate that this vU1 snRNA regulates expression of a subset of target genes at the level of pre-mRNA processing. This is the first indication that variant U1 snRNAs have a biological function in vivo. Furthermore, some vU1 snRNAs are packaged into unique ribonucleoproteins (RNPs), and many vU1 snRNA genes are differentially expressed in human embryonic stem cells (hESCs) and HeLa cells, suggesting developmental control of RNA processing through expression of different sets of vU1 snRNPs.

The carboxyl-terminal domain (CTD) of the largest subunit of RNA polymerase II (pol II) comprises multiple tandem repeats of the heptapeptide Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7. This unusual structure serves as a platform for the binding of factors required for expression of pol II-transcribed genes, including the small nuclear RNA (snRNA) gene-specific Integrator complex. The pol II CTD specifically mediates recruitment of Integrator to the promoter of snRNA genes to activate transcription and direct 3′ end processing of the transcripts. Phosphorylation of the CTD and a serine in position 7 are necessary for Integrator recruitment. Here, we have further investigated the requirement of the serines in the CTD heptapeptide and their phosphorylation for Integrator binding. We show that both Ser2 and Ser7 of the CTD are required and that phosphorylation of these residues is necessary and sufficient for efficient binding. Using synthetic phosphopeptides, we have determined the pattern of the minimal Ser2/Ser7 double phosphorylation mark required for Integrator to interact with the CTD. This novel double phosphorylation mark is a new addition to the functional repertoire of the CTD code and may be a specific signal for snRNA gene expression.

SummaryDrosha is the main RNase III-like enzyme involved in the process of microRNA (miRNA) biogenesis in the nucleus. Using whole-genome ChIP-on-chip analysis, we demonstrate that, in addition to miRNA sequences, Drosha specifically binds promoter-proximal regions of many human genes in a transcription-dependent manner. This binding is not associated with miRNA production or RNA cleavage. Drosha knockdown in HeLa cells downregulated nascent gene transcription, resulting in a reduction of polyadenylated mRNA produced from these gene regions. Furthermore, we show that this function of Drosha is dependent on its N-terminal protein-interaction domain, which associates with the RNA-binding protein CBP80 and RNA Polymerase II. Consequently, we uncover a previously unsuspected RNA cleavage-independent function of Drosha in the regulation of human gene expression.

RNA polymerase II transcribes both protein coding and non-coding RNA genes and, in yeast, different mechanisms terminate transcription of the two gene types. Transcription termination of mRNA genes is intricately coupled to cleavage and polyadenylation, whereas transcription of small nucleolar (sno)/small nuclear (sn)RNA genes is terminated by the RNA-binding proteins Nrd1, Nab3 and Sen1. The existence of an Nrd1-like pathway in humans has not yet been demonstrated. Using the U1 and U2 genes as models, we show that human snRNA genes are more similar to mRNA genes than yeast snRNA genes with respect to termination. The Integrator complex substitutes for the mRNA cleavage and polyadenylation specificity factor complex to promote cleavage and couple snRNA 3′-end processing with termination. Moreover, members of the associated with Pta1 (APT) and cleavage factor I/II complexes function as transcription terminators for human snRNA genes with little, if any, role in snRNA 3′-end processing. The gene-specific factor, proximal sequence element-binding transcription factor (PTF), helps clear the U1 and U2 genes of nucleosomes, which provides an easy passage for pol II, and the negative elongation factor facilitates termination at the end of the genes where nucleosome levels increase. Thus, human snRNA genes may use chromatin structure as an additional mechanism to promote efficient transcription termination in vivo.