Disassembly of interchromatin granule clusters alters the coordination of transcription and pre-mRNA splicing

To examine the involvement of interchromatin granule clusters (IGCs) in transcription and pre-mRNA splicing in mammalian cell nuclei, the serine-arginine (SR) protein kinase cdc2-like kinase (Clk)/STY was used as a tool to manipulate IGC integrity in vivo. Both immunofluorescence and transmission electron microscopy analyses of cells overexpressing Clk/STY indicate that IGC components are completely redistributed to a diffuse nuclear localization, leaving no residual structure. Conversely, overexpression of a catalytically inactive mutant, Clk/STY(K190R), causes retention of hypophosphorylated SR proteins in nuclear speckles. Our data suggest that the protein-protein interactions responsible for the clustering of interchromatin granules are disrupted when SR proteins are hyperphosphorylated and stabilized when SR proteins are hypophosphorylated. Interestingly, cells without intact IGCs continue to synthesize nascent transcripts. However, both the accumulation of splicing factors at sites of pre-mRNA synthesis as well as pre-mRNA splicing are dramatically reduced, demonstrating that IGC disassembly perturbs coordination between transcription and pre-mRNA splicing in mammalian cell nuclei

Proteomic analysis of interchromatin granule clusters

A variety of proteins involved in gene expression have been localized within mammalian cell nuclei in a speckled distribution that predominantly corresponds to interchromatin granule clusters (IGCs). We have applied a mass spectrometry strategy to identify the protein composition of this nuclear organelle purified from mouse liver nuclei. Using this approach, we have identified 146 proteins, many of which had already been shown to be localized to IGCs, or their functions are common to other already identified IGC proteins. In addition, we identified 32 proteins for which only sequence information is available and thus these represent novel IGC protein candidates. We find that 54% of the identified IGC proteins have known functions in pre-mRNA splicing. In combination with proteins involved in other steps of pre-mRNA processing, 81% of the identified IGC proteins are associated with RNA metabolism. In addition, proteins involved in transcription, as well as several other cellular functions, have been identified in the IGC fraction. However, the predominance of pre-mRNA processing factors supports the proposed role of IGCs as assembly, modification, and/or storage sites for proteins involved in pre-mRNA processing

Hypophosphorylated SR splicing factors transiently localize around active nucleolar organizing regions in telophase daughter nuclei

Upon completion of mitosis, daughter nuclei assemble all of the organelles necessary for the implementation of nuclear functions. We found that upon entry into daughter nuclei, snRNPs and SR proteins do not immediately colocalize in nuclear speckles. SR proteins accumulated in patches around active nucleolar organizing regions (NORs) that we refer to as NOR-associated patches (NAPs), whereas snRNPs were enriched at other nuclear regions. NAPs formed transiently, persisting for 15–20 min before dissipating as nuclear speckles began to form in G1. In the absence of RNA polymerase II transcription, NAPs increased in size and persisted for at least 2 h, with delayed localization of SR proteins to nuclear speckles. In addition, SR proteins in NAPs are hypophosphorylated, and the SR protein kinase Clk/STY colocalizes with SR proteins in NAPs, suggesting that phosphorylation releases SR proteins from NAPs and their initial target is transcription sites. This work demonstrates a previously unrecognized role of NAPs in splicing factor trafficking and nuclear speckle biogenesis

XE7: A novel splicing factor that interacts with ASF/SF2 and ZNF265

Pre-mRNA splicing is performed by the spliceosome. SR proteins in this macromolecular complex are essential for both constitutive and alternative splicing. By using the SR-related protein ZNF265 as bait in a yeast two-hybrid screen, we pulled out the uncharacterized human protein XE7, which is encoded by a pseudoautosomal gene. XE7 had been identified in a large-scale proteomic analysis of the human spliceosome. It consists of two different isoforms produced by alternative splicing. The arginine/serine (RS)-rich region in the larger of these suggests a role in mRNA processing. Herein we show for the first time that XE7 is an alternative splicing regulator. XE7 interacts with ZNF265, as well as with the essential SR protein ASF/SF2. The RS-rich region of XE7 dictates both interactions. We show that XE7 localizes in the nucleus of human cells, where it colocalizes with both ZNF265 and ASF/SF2, as well as with other SR proteins, in speckles. We also demonstrate that XE7 influences alternative splice site selection of pre-mRNAs from CD44, Tra2-β1 and SRp20 minigenes. We have thus shown that the spliceosomal component XE7 resembles an SR-related splicing protein, and can influence alternative splicing

Nuclear Organization and Dynamics of 7SK RNA in Regulating Gene Expression

We have identified 7SK RNA to be enriched in nuclear speckles. Knock-down of 7SK results in the mislocalization of nuclear speckle constituents, and the transcriptional up-regulation of a reporter gene locus. 7SK RNA transiently associates with the locus upon transcriptional down-regulation correlating with the displacement of pTEF-b

The Spectrum of C9orf72-mediated Neurodegeneration and Amyotrophic Lateral Sclerosis

The discovery that a hexanucleotide repeat expansion in C9orf72 is the most numerous genetic variant of both amyotrophic lateral sclerosis and frontotemporal dementia has opened a rapidly growing field, which may provide long hoped for advances in the understanding and treatment of these devastating diseases. In this review we describe the various phenotypes, clinical and pathological, associated with expansion of C9orf72, which go beyond amyotrophic lateral sclerosis and frontotemporal dementia to include neurodegeneration more broadly. Next we take a step back and summarize the current understanding of the C9orf72 expansion and its protein products at a molecular level. Three mechanisms are prominent: toxicity mediated directly by RNA transcribed from the repeat; toxicity mediated by dipeptide repeat proteins translated from the repeat sequence; and haploinsufficiency resulting from reduced transcription of the C9orf72 exonic sequence. A series of exciting advances have recently described how dipeptide repeat proteins might interfere with the normal role of the nucleolus in maturation of RNA binding proteins and in production of ribosomes. Importantly, these mechanisms are unlikely to be mutually exclusive. We draw attention to the fact that clinical and pathological similarities to other genetic variants without a repeat expansion must not be overlooked in ascribing a pathogenic mechanism to C9orf72-disease. Finally, with a view to impact on patient care, we discuss current practice with respect to genetic screening in patients with and without a family history of disease, and the most promising developments towards therapy that have been reported to date

Accurate Splicing of HDAC6 Pre-mRNA Requires SON

Pre-mRNA splicing requires proper splice site selection mediated by many factors including snRNPs and serine-arginine rich (SR) splicing factors. Our lab previously reported that the SR-like protein SON maintains organization of pre-mRNA splicing factors in nuclear speckles as well as splicing of many human transcripts including mRNAs coding for the chromatin-modifying enzymes HDAC6, ADA and SETD8. However, the mechanism by which SON maintains accurate splicing is unknown. To build tools for understanding SON-dependent pre-mRNA splicing, we constructed a minigene reporter plasmid driving expression of the genomic sequence spanning exons 26 through 29 of HDAC6. Following SON depletion, we observed altered splicing of HDAC6 reporter transcripts that showed exclusion of exons 27 and 28, reflecting the splicing patterns of endogenous HDAC6 mRNA. Importantly, loss of HDAC6 biological function was also observed, as indicated by truncated HDAC6 protein and corresponding absence of aggresome assembly activities of HDAC6 binding-of-ubiquitin zinc finger (BUZ) domain. We therefore propose that SON-mediated splicing regulation of HDAC6 is essential for supporting protein degradation pathways that prevent human disease

Alignment of Mitotic Chromosomes in Human Cells Involves SR-Like Splicing Factors Btf and TRAP150

Serine-arginine-rich (SR) or SR-like splicing factors interact with exon junction complex proteins during pre-mRNA processing to promote mRNA packaging into mature messenger ribonucleoproteins (mRNPs) and to dictate mRNA stability, nuclear export, and translation. The SR protein family is complex, and while many classical SR proteins have well-defined mRNA processing functions, those of other SR-like proteins is unclear. Here, we show that depletion of the homologous non-classical serine-arginine-rich (SR) splicing factors Bcl2-associated transcription factor (Btf or BCLAF) and thyroid hormone receptor-associated protein of 150 kDa (TRAP150) causes mitotic defects. We hypothesized that the depletion of these SR-like factors affects mitosis indirectly through an altered expression of mitotic checkpoint regulator transcripts. We observed an altered abundance of transcripts that encode mitotic regulators and mitotic chromosome misalignment defects following Btf and/or TRAP150 depletion. We propose that, in addition to their previously reported roles in maintaining mRNA distribution, Btf and TRAP150 control the abundance of transcripts encoding mitotic regulators, thereby affecting mitotic progression in human cells