Project description:Nuclear speckles, a type of membraneless nuclear organelle in higher eukaryotic cells, play a vital role in gene expression regulation. Using the reverse transcription-based RBP binding sites sequencing (ARTR-seq) method, we study human transcripts associated with nuclear speckles. We identify three gene groups whose transcripts demonstrate different speckle localization properties and dynamics – stably enriched in nuclear speckle post-transcriptionally, transiently enriched in speckles at the pre-mRNA stage co-transcriptionally, and not enriched in speckles. We show that nuclear speckles specifically facilitate splicing of speckle-enriched transcripts. We further reveal RNA sequence features contributing to transcript speckle localization, underscoring a tight interplay between genome organization, RNA cis-elements, and transcript speckle enrichment, and connecting transcript speckle localization with splicing logic. Finally, we show that speckles can act as hubs for the regulated retention of introns during cellular stress. Collectively, our data highlight a role of nuclear speckles in both co- and post-transcriptional splicing regulation.
Project description:Nuclear speckles, a type of membraneless nuclear organelle in higher eukaryotic cells, play a vital role in gene expression regulation. Using the reverse transcription-based RBP binding sites sequencing (ARTR-seq) method, we study human transcripts associated with nuclear speckles. We identify three gene groups whose transcripts demonstrate different speckle localization properties and dynamics – stably enriched in nuclear speckle post-transcriptionally, transiently enriched in speckles at the pre-mRNA stage co-transcriptionally, and not enriched in speckles. We show that nuclear speckles specifically facilitate splicing of speckle-enriched transcripts. We further reveal RNA sequence features contributing to transcript speckle localization, underscoring a tight interplay between genome organization, RNA cis-elements, and transcript speckle enrichment, and connecting transcript speckle localization with splicing logic. Finally, we show that speckles can act as hubs for the regulated retention of introns during cellular stress. Collectively, our data highlight a role of nuclear speckles in both co- and post-transcriptional splicing regulation.
Project description:MALAT-1 is a long mammalian non-coding transcript of approximately 8500 nucleotides. It is localized to nuclear speckles despite its mRNA-like characteristics, which usually result in the export of transcripts to the cytoplasm. In the present study, we report the identification of several factors that influence the nuclear speckle localization of MALAT-1, and we provide evidence that MALAT-1 is involved in the regulation of gene expression. Heterokaryon assays revealed that MALAT-1 does not shuttle between the nucleus and cytoplasm, but is stably retained within the nucleus. Analysis of MALAT-1 fragments showed that MALAT-1 contains two distinct nuclear speckle-directing elements (between nucleotides 1961 to 3040 and 6008 to 7011 of the MALAT-1 sequence). The knockdown of the nuclear speckle proteins, RNPS1, SRm160 or IBP160, resulted in the diffusion of MALAT-1 to the nucleoplasm. In addition, we have demonstrated that depletion of MALAT-1 represses the expression of several genes. This repression of gene expression also occurs in response to the delocalization of MALAT-1 from the nuclear speckles. These results suggest that RNPS1, SRm160 and IBP160 contribute to the localization of MALAT-1 to nuclear speckles where it is involved in regulating gene expression
Project description:Nuclear noncoding RNAs are key regulators of gene expression and chromatin organization. Here we present RedChIP, a technique combining RNA-DNA proximity ligation and chromatin immunoprecipitation for identification of RNAs associated with genomic regions occupied by a protein of interest. We identify a spectrum of cis and trans acting lincRNAs enriched at Polycomb and CTCF binding sites, which may be involved in Polycomb mediated gene repression and chromatin looping.
Project description:Gene regulation occurs through trans-acting factors (e.g. transcription factors) acting on cis-regulatory elements (e.g. enhancers). Massively parallel reporter assays (MPRAs) functionally survey large numbers of cis-regulatory elements for regulatory potential, but do not identify the trans-acting factors that mediate any observed effects. Here we include preliminary data from a pilot transMPRA experiment — a reporter assay that efficiently combines multiplex CRISPR-mediated perturbation and MPRAs to identify trans-acting factors that modulate the regulatory activity of specific enhancers.
Project description:The nucleus is highly organized such that factors involved in transcription and processing of distinct classes of RNA are organized within specific nuclear bodies. One example is the nuclear speckle, which is defined by high concentrations of protein and non-coding RNA regulators of pre-mRNA splicing. What functional role, if any, speckles might play in the process of mRNA splicing remains unknown. Here we show that genes localized near nuclear speckles display higher spliceosome concentrations, increased spliceosome binding to their pre-mRNAs, and higher co-transcriptional splicing levels relative to genes that are located farther from nuclear speckles. We show that directed recruitment of a pre-mRNA to nuclear speckles is sufficient to increase mRNA splicing levels. Finally, we show that gene organization around nuclear speckles is highly dynamic with differential localization between cell types corresponding to differences in splicing efficiency. Together, our results integrate the longstanding observations of nuclear speckles with the biochemistry of mRNA splicing and demonstrate a critical role for dynamic 3D spatial organization of genomic DNA in driving spliceosome concentrations and controlling the efficiency of mRNA splicing.
Project description:Transcription is controlled by the interactions of cis-acting DNA elements with diffusible trans-acting factors. Changes in cis or trans factors can drive expression divergence within and between species, and the relative prevalence of each can reveal the evolutionary path to variation. Previous work delineating the mode of expression divergence in animals has largely used whole body expression measurements in a single condition. Since cis-acting elements often drive expression in a subset of cell types or conditions, these measurements may not capture the complete contribution of cis-acting changes. Here, we quantify the mode of expression divergence in the Drosophila fat body, the primary immune organ, in several conditions. We performed allele-specific expression analysis using two geographically distinct lines of D. melanogaster and their F1 hybrids. We performed separate infections with Gram-negative S. marcescens or Gram-positive E. faecalis bacteria, which trigger the two primary signaling pathways in the Drosophila innate immune response. The mode of expression divergence strongly depends on the condition, with trans-acting effects dominating in response to Gram-positive infection and cis-acting effects dominating in Gram-negative and pre-infection conditions. Expression divergence in several receptor proteins may underlie the infection-specific trans effects. Before infection, when the fat body has a metabolic role, there are many compensatory effects, changes in cis and trans that counteract each other to maintain expression levels. This work demonstrates that within a single tissue, the mode of expression divergence varies between conditions and suggests that these differences reflect the diverse evolutionary histories of host-pathogen interactions.
Project description:Background: Expression QTL analyses have shed light on transcriptional regulation in numerous species of plants, animals, and yeasts. These microarray-based analyses identify regulators of gene expression as either cis-acting factors that regulate proximal genes, or trans-acting factors that function through a variety of mechanisms to affect transcript abundance of unlinked genes. Results: A hydroponics-based genetical genomics study in roots of a Zea mays IBM2 Syn10 double haploid population identified tens of thousands of cis-acting and trans-acting eQTL. Cases of false-positive eQTL, which results from the lack of complete genomic sequences from both parental genomes, were described. A candidate gene for a trans-acting regulatory factor was identified through positional cloning. The unexpected regulatory function of a class I glutamine amidotransferase controls the expression of an ABA 8’-hydroxylase pseudogene.