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.

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