Posttranscriptional modifications such as alternative pre-mRNA splicing and RNA editing, as well as posttranslational modifications of proteins, including phosphorylation, sumoylation, and ubiquitylation, can increase the proteome size, but this is unlikely to fully account for the complexity associated with higher eukaryotes (reviewed in Prasanth and Spector, 2007). Recent large-scale genomic sequencing analyses have revealed that human and mouse bear very similar number of protein coding genes (∼23,000), surprisingly this number is not very different from Caenorhabditis elegans (∼20,000) or Drosophila (∼14,000) ( This suggests that the complexity associated with higher organisms is not solely related to the number of protein coding genes that they harbor (reviewed in Mattick, 2001 Frith et al., 2005 Mattick and Makunin, 2006 Kapranov et al., 2007 Pheasant and Mattick, 2007 Prasanth and Spector, 2007 Wilusz et al., 2009).
In addition to their protein constituents, some of these subnuclear domains also harbor RNA molecules (reviewed in Prasanth and Spector, 2007 Wilusz et al., 2009). However, the proteins in these domains generally show high levels of exchange between the domains and the surrounding interchromatin space (reviewed in Misteli, 2001 Misteli, 2008).
The majority of nuclear bodies show little (PML bodies and Cajal bodies) to no mobility in the nucleus ( Muratani et al., 2002 Platani et al., 2002). Nuclear bodies are broadly characterized by their macromolecular composition, number, size, and associated functions. To efficiently carry out these functions, the mammalian cell nucleus is compartmentalized into specific subnuclear domains or nuclear bodies (reviewed in Spector, 2006 Matera et al., 2009). The eukaryotic cell nucleus is a membrane-bounded organelle where many critical cellular functions are carried out within macromolecular protein complexes (reviewed in Prasanth and Spector, 2005). Our results suggest that 7SK RNA plays a role in modulating the available level of P-TEFb upon transcriptional down-regulation by sequestering its constituents in nuclear speckles. Furthermore, 7SK RNA transiently associates with a stably integrated reporter gene locus upon transcriptional down-regulation and its presence correlates with the efficient displacement of P-TEFb constituents from the locus.
Our results demonstrate that knock-down of 7SK RNA, by specific antisense oligonucleotides, results in the mislocalization of nuclear speckle constituents in a transcription-dependent manner, and the transcriptional up-regulation of a RNA polymerase II transcribed reporter gene locus. Inhibition occurs via sequestration of the active P-TEFb kinase complex (CDK 9 and Cyclin T1/T2a/b or K) that is involved in phosphorylating the C-terminal domain of RNA polymerase II. 7SK RNA, in association with HEXIM 1 and 2, is involved in the inhibition of transcriptional elongation by RNA polymerase II. We have identified 7SK RNA to be enriched in nuclear speckles or interchromatin granule clusters (IGCs), a subnuclear domain enriched in pre-mRNA processing factors. Noncoding RNAs play important roles in various aspects of gene regulation.
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