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.

• MeSH
• antisense oligodeoxyribonukleotidy genetika   metabolismus   MeSH
• HeLa buňky MeSH
• lidé MeSH
• messenger RNA chemie   metabolismus   MeSH
• mikroinjekce MeSH
• mutace MeSH
• prekurzory RNA chemie   metabolismus   MeSH
• sestřih RNA * MeSH
• spliceozomy genetika   metabolismus   MeSH
• struktury buněčného jádra genetika   metabolismus   ultrastruktura   MeSH
• teplota MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• antisense oligodeoxyribonukleotidy MeSH
• messenger RNA MeSH
• prekurzory RNA MeSH

In the present study, the spatial organization of intron-containing pre-mRNAs of Epstein-Barr virus (EBV) genes relative to location of splicing factors is investigated. The intranuclear position of transcriptionally active EBV genes, as well as of nascent transcripts, is found to be random with respect to the speckled accumulations of splicing factors (SC35 domains) in Namalwa cells, arguing against the concept of the locus-specific organization of mRNA genes with respect to the speckles. Microclusters of splicing factors are, however, frequently superimposed on nascent transcript sites. The transcript environment is a dynamic structure consisting of both nascent and released transcripts, i.e., the track-like transcript environment. Both EBV sequences of the chromosome 1 homologue are usually associated with the track, are transcriptionally active, and exhibit in most cases a polar orientation. In contrast to nascent transcripts (in the form of spots), the association of a post-transcriptional pool of viral pre-mRNA (in the form of tracks) with speckles is not random and is further enhanced in transcriptionally silent cells when splicing factors are sequestered in enlarged accumulations. The transcript environment reflects the intranuclear transport of RNA from the sites of transcription to SC35 domains, as shown by concomitant mapping of DNA, RNA, and splicing factors. No clear vectorial intranuclear trafficking of transcripts from the site of synthesis toward the nuclear envelope for export into the cytoplasm is observed. Using Namalwa and Raji cell lines, a correlation between the level of viral gene transcription and splicing factor accumulation within the viral transcript environment has been observed. This supports a concept that the level of transcription can alter the spatial relationship among intron-containing genes, their transcripts, and speckles attributable to various levels of splicing factors recruited from splicing factor reservoirs. Electron microscopic in situ hybridization studies reveal that the released transcripts are directed toward reservoirs of splicing factors organized in clusters of interchromatin granules. Our results point to the bidirectional intranuclear movement of macromolecular complexes between intron-containing genes and splicing factor reservoirs: the recruitment of splicing factors to transcription sites and movement of released transcripts from DNA loci to reservoirs of splicing factors.

• MeSH
• biologický transport MeSH
• buněčné jádro genetika   metabolismus   ultrastruktura   virologie   MeSH
• DNA řízené RNA-polymerasy antagonisté a inhibitory   metabolismus   MeSH
• DNA virů genetika   metabolismus   MeSH
• elektronová mikroskopie MeSH
• fluorescenční mikroskopie MeSH
• genetická transkripce genetika   MeSH
• genom virový MeSH
• heterogenní jaderné ribonukleoproteiny MeSH
• introny genetika   MeSH
• jaderné proteiny metabolismus   MeSH
• konfokální mikroskopie MeSH
• lidé MeSH
• messenger RNA genetika   metabolismus   MeSH
• nádorové buňky kultivované MeSH
• plazmidy genetika   MeSH
• prekurzory RNA genetika   metabolismus   MeSH
• ribonukleoproteiny metabolismus   MeSH
• RNA virová genetika   metabolismus   MeSH
• serin-arginin sestřihové faktory MeSH
• spliceozomy genetika   metabolismus   ultrastruktura   MeSH
• virové geny genetika   MeSH
• virus Epsteinův-Barrové genetika   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DNA řízené RNA-polymerasy MeSH
• DNA virů MeSH
• heterogenní jaderné ribonukleoproteiny MeSH
• jaderné proteiny MeSH
• messenger RNA MeSH
• prekurzory RNA MeSH
• ribonukleoproteiny MeSH
• RNA virová MeSH
• serin-arginin sestřihové faktory MeSH
• SRSF2 protein, human MeSH Prohlížeč