Malat1 is an abundant long, noncoding RNA that localizes to nuclear bodies known as nuclear speckles, which contain a distinct set of pre-mRNA processing factors. Previous studies in cell culture have demonstrated that Malat1 interacts with pre-mRNA splicing factors, including the serine-and arginine-rich (SR) family of proteins, and regulates a variety of biological processes, including cancer cell migration, synapse formation, cell cycle progression, and responses to serum stimulation. To address the physiological function of Malat1 in a living organism, we generated Malat1-knockout (KO) mice using homologous recombination. Unexpectedly, the Malat1-KO mice were viable and fertile, showing no apparent phenotypes. Nuclear speckle markers were also correctly localized in cells that lacked Malat1. However, the cellular levels of another long, noncoding RNA-Neat1-which is an architectural component of nuclear bodies known as paraspeckles, were down-regulated in a particular set of tissues and cells lacking Malat1. We propose that Malat1 is not essential in living mice maintained under normal laboratory conditions and that its function becomes apparent only in specific cell types and under particular conditions.
The rate of RNA polymerase II (Pol II) elongation can influence splice site selection in nascent transcripts, yet the extent and physiological relevance of this kinetic coupling between transcription and alternative splicing (AS) is not well understood. We performed experiments to perturb Pol II elongation and then globally compared AS patterns with genome-wide Pol II occupancy. RNA binding and RNA processing functions were significantly enriched among the genes with Pol II elongation inhibition-dependent changes in AS. Under conditions that interfere with Pol II elongation, including cell stress, increased Pol II occupancy was detected in the intronic regions flanking the alternative exons in these genes, and these exons generally became more included. A disproportionately high fraction of these exons introduced premature termination codons that elicited nonsense-mediated mRNA decay (NMD), thereby further reducing transcript levels. Our results provide evidence that kinetic coupling between transcription, AS, and NMD affords a rapid mechanism by which cells can respond to changes in growth conditions, including cell stress, to coordinate the levels of RNA processing factors with mRNA levels.
The role of alternative splicing (AS) in eliciting immune responses is poorly understood. We used quantitative AS microarray profiling to survey changes in AS during activation of Jurkat cells, a leukemia-derived T-cell line. Our results indicate that ;10%-15% of the profiled alternative exons undergo a >10% change in inclusion level during activation. The majority of the genes displaying differential AS levels are distinct from the set of genes displaying differential transcript levels. These two gene sets also have overlapping yet distinct functional roles. For example, genes that show differential AS patterns during T-cell activation are often closely associated with cell-cycle regulation, whereas genes with differential transcript levels are highly enriched in functions associated more directly with immune defense and cytoskeletal architecture. Previously unknown AS events were detected in genes that have important roles in T-cell activation, and these AS level changes were also observed during the activation of normal human peripheral CD4+ and CD8+ lymphocytes. In summary, by using AS microarray profiling, we have discovered many new AS changes associated with T-cell activation. Our results suggest an extensive role for AS in the regulation of the mammalian immune response.
A key challenge in understanding and ultimately treating autism is to identify common molecular mechanisms underlying this genetically heterogeneous disorder. Transcriptomic profiling of autistic brains has revealed correlated misregulation of the neuronal splicing regulator nSR100/SRRM4 and its target microexon splicing program in more than one-third of analyzed individuals. To investigate whether nSR100 misregulation is causally linked to autism, we generated mutant mice with reduced levels of this protein and its target splicing program. Remarkably, these mice display multiple autistic-like features, including altered social behaviors, synaptic density, and signaling. Moreover, increased neuronal activity, which is often associated with autism, results in a rapid decrease in nSR100 and splicing of microexons that significantly overlap those misregulated in autistic brains. Collectively, our results provide evidence that misregulation of an nSR100-dependent splicing network controlled by changes in neuronal activity is causally linked to a substantial fraction of autism cases.
In a recent study, we provided evidence that strong promoter-bound transcriptional activators result in higher levels of splicing and 3-end cleavage of nascent pre-mRNA than do weak promoter-bound activators and that this effect of strong activators requires the carboxyl-terminal domain (CTD) of RNA polymerase II (pol II). In the present study, we have investigated the mechanism of activator-and CTD-mediated stimulation of pre-mRNA processing. Affinity chromatography experiments reveal that two factors previously implicated in the coupling of transcription and pre-mRNA processing, PSF and p54 nrb /NonO, preferentially bind a strong rather than a weak activation domain. Elevated expression in human 293 cells of PSF bypasses the requirement for a strong activator to promote efficient splicing and 3-end cleavage. Truncation of the pol II CTD, which consists of 52 repeats of the consensus heptapeptide sequence YSPTSPS, to 15 heptapeptide repeats prevents PSF-dependent stimulation of splicing and 3-end cleavage. Moreover, PSF and p54 nrb /NonO bind in vitro to the wild-type CTD but not to the truncated 15-repeat CTD, and domains in PSF that are required for binding to activators and to the CTD are also important for the stimulation of pre-mRNA processing. Interestingly, activator-and CTD-dependent stimulation of splicing mediated by PSF appears to primarily affect the removal of first introns. Collectively, these results suggest that the recruitment of PSF to activated promoters and the pol II CTD provides a mechanism by which transcription and pre-mRNA processing are coordinated within the cell.The processing of precursor mRNA (pre-mRNA) to mRNA in the nucleus of eukaryotic cells occurs by a series of coordinated steps that include the addition of a 5Ј-end m 7 G cap, splicing, and 3Ј-end formation. In most cases, 3Ј-end formation involves an endonucleolytic cleavage reaction downstream of an AAUAAA signal sequence, followed by the addition of a poly(A) tail (11,68). Each of these processing steps can occur independently of one another in vitro, although in vivo, increasing evidence indicates that there is extensive communication between factors that participate in each of the processing reactions (28,35,41,52). In some cases, one step can modulate the efficiency of another. Currently, little is known about the specific mechanisms by which different steps in the maturation of mRNA regulate each other.Transcription is initiated within the context of a multisubunit holoenzyme complex that contains general transcription factors and RNA polymerase II (pol II) (26, 37). Sequence-specific DNA binding transcriptional activators play an important role in the recruitment of the pol II holoenzyme complex to promoter regions (53). RNA pol II contains a unique C-terminal domain (CTD) that consists in mammalian cells of 52 repeats of the heptapeptide consensus sequence YSPTSPS (2, 12). The CTD is important for the response of certain genes to promoter-bound activators (1,25,40,60), although activators can also stimulate transcrip...
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