Inefficient processing impairs release of RNA from the site of transcription

Custódio, Noélia; Carmo‐Fonseca, Maria; Geraghty, Finola; Pereira, H. Sofia; Grosveld, Frank; Antoniou, Michael

doi:10.1093/emboj/18.10.2855

Cited by 196 publications

(218 citation statements)

References 65 publications

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“…The inhibitor flavopiridol, which we used above, cannot help distinguish between these possibilities because its application results in the loss of transcribing RNA polymerase (Bensaude, 2011;Jonkers, Kwak and Lis, 2014). Here, we applied a different inhibitor, actinomycin-D (Act-D), which arrests RNA polymerases during transcription (Custódio et al, 1999;Bensaude, 2011). The arrested polymerases are temporarily retained along with their associated transcripts, which can be visualized as RNA foci (Custódio et al, 1999).…”

Section: Transcriptional Activity and Rna Play Distinct Roles In Euchmentioning

confidence: 99%

Transcription organizes euchromatin similar to an active microemulsion

Hilbert

Sato

Kimurâ

et al. 2017

Preprint

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Graphical abstract HighlightsThe copyright holder for this preprint (which was . http://dx.doi.org/10.1101/234112 doi: bioRxiv preprint first posted online Dec. 15, 2017; Page 2 of 46 SummaryThe three-dimensional organization of the genome is essential for development and health. Although the organization of euchromatin (transcriptionally permissive chromatin) dynamically adjusts to changes in transcription, the underlying mechanisms remain elusive. Here, we studied how transcription organizes euchromatin, using experiments in zebrafish embryonic cells and theory. We show that transcription establishes an interspersed pattern of chromatin domains and RNA-enriched microenvironments. Specifically, accumulation of RNA in the vicinity of transcription sites creates microenvironments that locally remodel chromatin by displacing not transcribed chromatin. Ongoing transcriptional activity stabilizes the interspersed pattern of chromatin domains and RNA-enriched microenvironments by establishing contacts between chromatin and RNA. We explain our observations with an active microemulsion model based on two macromolecular mechanisms: RNA/RNA-binding protein complexes generally segregate from chromatin, while transcribed chromatin is retained among RNA/RNA-binding protein accumulations. We propose that microenvironments might be central to genome architecture and serve as gene regulatory hubs.

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Section: Transcriptional Activity and Rna Play Distinct Roles In Euchmentioning

confidence: 99%

Transcription organizes euchromatin similar to an active microemulsion

Hilbert

Sato

Kimurâ

et al. 2017

Preprint

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“…The human PABPN1 transgene was expressed under the control of the human Des-LCR and promoter 31 ( Figure 1A), and mRNA was stabilized with the use of the 3= half of the HBB gene. [43][44][45][46] Stable clones of ImmortoMouse myoblasts (clone IM2) 33 were generated, and the transgene mRNA expression level was determined using RT-qPCR. The D7E and WTA clones, expressing the Ala17 or Ala10 alleles, respectively (Figure 1, A and B), were selected for further study.…”

Section: Expression Of Wt or Exppabpn1 In Mouse Myotube Cultures At Lmentioning

confidence: 99%

Modeling Oculopharyngeal Muscular Dystrophy in Myotube Cultures Reveals Reduced Accumulation of Soluble Mutant PABPN1 Protein

Raz

Routledge

Venema

et al. 2011

The American Journal of Pathology

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Oculopharyngeal muscular dystrophy (OPMD) is an autosomal dominant disease caused by an alanine tract expansion mutation in poly(A) binding protein nuclear 1 (expPABPN1). To model OPMD in a myogenic and physiological context, we generated mouse myoblast cell clones stably expressing either human wild type (WT) or expPABPN1 at low levels. Transgene expression is induced on myotube differentiation and results in formation of insoluble nuclear PABPN1 aggregates that are similar to those observed in patients with OPMD. Quantitative analysis of PABPN1 in myotube cultures revealed that expPABPN1 accumulation and aggregation is greater than that of the WT protein. We found that aggregation of expPABPN1 is more affected than WT PABPN1 by inhibition of proteasome activity. Consistent with this, in myotube cultures expressing expPABPN1, deregulation of the proteasome was identified as the most significantly perturbed pathway. Differences in the accumulation of soluble WT and expPABPN1 were consistent with differences in ubiquitination and rate of protein turnover. This study demonstrates, for the first time to our knowledge, that, in myotubes, the ratio of soluble/insoluble expPABPN1 is significantly lower compared with that of the WT protein. We suggest that this difference can contribute to muscle weakness in OPMD.

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“…The splicing of pre-mRNAs may be a cotranscriptional event (Beyer and Osheim, 1988;Neugebauer and Roth, 1997;Custodio et al, 1999). However, not all pre-mRNA sequences are processed cotranscriptionally and posttranscriptional splicing does occur (Zachar et al, 1993;Baurén and Wieslander, 1994;Wuarin and Schibler, 1994).…”

confidence: 99%

“…Spliceosomes are generated by the constitutive assembly of U1, U2, U5, U4/U6 small nuclear ribonucleoprotein particles (snRNPs) and various non-snRNP factors on pre-mRNAs in the cascade of sequence-specific steps (Steitz et al, 1988;Lamm and Lamond, 1993;Moore et al, 1993;Newman, 1994;Krämer, 1996). The formation of the functional spliceosome is thus preceded by the formation of a number of prespliceosomal complexes, termed E, A, and B complexes containing, together with unspliced pre-mRNA, defined combinations of snRNP particles and non-snRNP factors (Steitz et al, 1988;Lamm and Lamond, 1993;Moore et al, 1993;Newman, 1994;Krämer, 1996).The splicing of pre-mRNAs may be a cotranscriptional event (Beyer and Osheim, 1988;Neugebauer and Roth, 1997;Custodio et al, 1999). However, not all pre-mRNA sequences are processed cotranscriptionally and posttranscriptional splicing does occur (Zachar et al, 1993;Baurén and Wieslander, 1994;Wuarin and Schibler, 1994).…”

mentioning

confidence: 99%

Prespliceosomal Assembly on Microinjected Precursor mRNA Takes Place in Nuclear Speckles

Melčák

Kopský

et al. 2001

MBoC

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Nuclear speckles (speckles) represent a distinct nuclear compartment within the interchromatin space and are enriched in splicing factors. They have been shown to serve neighboring active genes as a reservoir of these factors. In this study, we show that, in HeLa cells, the (pre)spliceosomal assembly on precursor mRNA (pre-mRNA) is associated with the speckles. For this purpose, we used microinjection of splicing competent and mutant adenovirus pre-mRNAs with differential splicing factor binding, which form different (pre)spliceosomal complexes and followed their sites of accumulation. Splicing competent pre-mRNAs are rapidly targeted into the speckles, but the targeting is temperature-dependent. The polypyrimidine tract sequence is required for targeting, but, in itself, is not sufficient. The downstream flanking sequences are particularly important for the targeting of the mutant pre-mRNAs into the speckles. In supportive experiments, the behavior of the speckles was followed after the microinjection of antisense deoxyoligoribonucleotides complementary to the specific domains of snRNAs. Under these latter conditions prespliceosomal complexes are formed on endogenous pre-mRNAs. We conclude that the (pre)spliceosomal complexes on microinjected pre-mRNA are formed inside the speckles. Their targeting into and accumulation in the speckles is a result of the cumulative loading of splicing factors to the pre-mRNA and the complexes formed give rise to the speckled pattern observed. INTRODUCTIONNuclear speckles are enriched in splicing factors and in the factors of the transcription machinery (Spector, 1990;Krause et al., 1994;Bregman et al., 1995;Larsson et al., 1995). Even though there is a consensus that these compartments play a role in RNA metabolism, their exact function is presently unknown. When growing mammalian cells are labeled with antibodies to splicing components, 20 to 50 shining nuclear domains, i.e., nuclear speckles, also termed SC35 domains (protein SC35 being an important serine/arginine (SR)-rich splicing factor [Fu and Maniatis, 1990]), splicing factor compartments, or just speckles, are usually observed (Perraud et al., 1979;Spector et al., 1983;Spector, 1990;Misteli, 2000). In some cell types, they occupy as much as 20% of the nuclear volume. At the electron microscope level, they consist of morphologically well-defined interchromatin granule clusters and of domains of perichromatin fibrils, some of which are believed to represent precursor-mRNAs (premRNAs) (Fakan and Puvion, 1980;Spector et al., 1983;Puvion et al., 1984;Spector, 1990;Fakan, 1994;Raška, 1995;Melčák et al., 2000).Most mammalian pre-mRNAs contain introns and typically have to be spliced before being transported to the cytoplasm. It has been shown biochemically that spliceosome formation and splicing may be cotranscriptional events (Wuarin and Schibler, 1994). More recent results indicate that transcription and splicing are coupled with interactions of certain factors in both processes. They take part in the large macromolecular c...

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Inefficient processing impairs release of RNA from the site of transcription

Cited by 196 publications

References 65 publications

Transcription organizes euchromatin similar to an active microemulsion

Transcription organizes euchromatin similar to an active microemulsion

Modeling Oculopharyngeal Muscular Dystrophy in Myotube Cultures Reveals Reduced Accumulation of Soluble Mutant PABPN1 Protein

Prespliceosomal Assembly on Microinjected Precursor mRNA Takes Place in Nuclear Speckles

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