Prespliceosomal Assembly on Microinjected Precursor mRNA Takes Place in Nuclear Speckles

Abstract

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...

A role for TREX components in the release of spliced mRNA from nuclear speckle domains

“…A great deal of elegant work has dealt with these issues and also with the detailed morphology of nuclear speckle domains and sub-domains within nuclear speckle domains 5, 44 (and refs therein). In addition, previous work 17 and our study show that nuclear speckle domains often appear enlarged and/or coalesced, possibly because of the large amount of RNA associated with the domains under certain conditions. Our study is not focused on these detailed aspects of nuclear speckle domain formation, dynamics, and morphology, and thus, with these caveats in mind, for simplicity, we will refer to transcripts as being in nuclear speckle domains throughout this manuscript.…”

Influenza virus mRNA trafficking through host nuclear speckles

“…Although controversial, it has been reported that post-transcription splicing may be spatially mediated at nuclear speckles 6 . This concept is supported by accumulation of specific polyadenylated mRNAs at nuclear speckles, including mRNA originated from plasmid, which appear to be involved in splicing and/or export of these mRNAs to the cytoplasm 7–10 . However, most cellular pre-mRNAs do not utilize nuclear speckles, which serve as suppliers of factors with key roles in gene expression 1 .…”

TDP-43 localizes in mRNA transcription and processing sites in mammalian neurons

“…In addition to chromosome territories (Cremer and Cremer, 2001), other well characterized nuclear compartments include the nucleolus, the site of synthesis and processing of rRNAs (for review, see Raska et al, 2006;Boisvert et al, 2007), the Cajal body that participates in the biogenesis of small nuclear and nucleolar ribonucleoproteins (snRNPs and snoRNPs; Gall, 2000;Cioce and Lamond, 2005), and the nuclear speckles, which act as splicing factor reservoirs (for review, see Raska, 1995;Misteli et al, 1997;Lamond and Spector, 2003) and sites of trafficking and post-transcriptional processing of pre-mRNAs (Melcák et al, 2000(Melcák et al, , 2001. All steps of RNA transcription and nuclear processing require the formation of RNP complexes, which play important roles in mRNA surveillance and function (Moore, 2005).…”