Serine-arginine-rich (SR) proteins play a key role in alternative pre-mRNA splicing in eukaryotes. We recently showed that a large SR protein called Son has unique repeat motifs that are essential for maintaining the subnuclear organization of pre-mRNA processing factors in nuclear speckles. Motif analysis of Son highlights putative RNA interaction domains that suggest a direct role for Son in pre-mRNA splicing. Here, we used in situ approaches to show that Son localizes to a reporter minigene transcription site, and that RNAi-mediated Son depletion causes exon skipping on reporter transcripts at this transcription site. A genome-wide exon microarray analysis was performed to identify human transcription and splicing targets of Son. Our data show that Son-regulated splicing encompasses all known types of alternative splicing, the most common being alternative splicing of cassette exons. We confirmed that knockdown of Son leads to exon skipping in pre-mRNAs for chromatin-modifying enzymes, including ADA, HDAC6 and SetD8. This study reports a comprehensive view of human transcription and splicing targets for Son in fundamental cellular pathways such as integrin-mediated cell adhesion, cell cycle regulation, cholesterol biosynthesis, apoptosis and epigenetic regulation of gene expression.

The cytoplasm of the eukaryotic cell is subdivided into distinct functional domains by the presence of a variety of membrane-bound organelles. The remaining aqueous space may be further partitioned by the regulated assembly of discrete ribonucleoprotein (RNP) complexes that contain particular proteins and messenger RNAs. These RNP granules are conserved structures whose importance is highlighted by studies linking them to human disorders like amyotrophic lateral sclerosis. However, relatively little is known about the diversity, composition, and physiological roles of these cytoplasmic structures. To begin to address these issues, we examined the cytoplasmic granules formed by a key set of signaling molecules, the protein kinases of the budding yeast Saccharomyces cerevisiae. Interestingly, a significant fraction of these proteins, almost 20%, was recruited to cytoplasmic foci specifically as cells entered into the G 0 -like quiescent state, stationary phase. Colocalization studies demonstrated that these foci corresponded to eight different granules, including four that had not been reported previously. All of these granules were found to rapidly disassemble upon the resumption of growth, and the presence of each was correlated with cell viability in the quiescent cultures. Finally, this work also identified new constituents of known RNP granules, including the well-characterized processing body and stress granule. The composition of these latter structures is therefore more varied than previously thought and could be an indicator of additional biological activities being associated with these complexes. Altogether, these observations indicate that quiescent yeast cells contain multiple distinct cytoplasmic granules that may make important contributions to their long-term survival.

The interior of the eukaryotic cell is a highly compartmentalized space containing both membrane-bound organelles and the recently identified nonmembranous ribonucleoprotein (RNP) granules. This study examines in Saccharomyces cerevisiae the assembly of one conserved type of the latter compartment, known as the stress granule. Stress granules form in response to particular environmental cues and have been linked to a variety of human diseases, including amyotrophic lateral sclerosis. To further our understanding of these structures, a candidate genetic screen was employed to identify regulators of stress granule assembly in quiescent cells. These studies identified a ubiquitin-specific protease, Ubp3, as having an essential role in the assembly of these RNP granules. This function was not shared by other members of the Ubp protease family and required Ubp3 catalytic activity as well as its interaction with the cofactor Bre5. Interestingly, the loss of stress granules was correlated with a decrease in the long-term survival of stationary-phase cells. This phenotype is similar to that observed in mutants defective for the formation of a related RNP complex, the Processing body. Altogether, these observations raise the interesting possibility of a general role for these types of cytoplasmic RNP granules in the survival of G 0 -like resting cells.T he interior of the eukaryotic cell is a highly compartmentalized space. Organelles harbor distinct sets of proteins that often possess related functions, serving to organize the cellular milieu and increase reaction efficiencies. The recent identification of a family of ribonucleoprotein (RNP) granules suggests that this complexity may be greater than previously recognized (1, 2). These RNP granules differ from traditional organelles in that they lack a limiting membrane and form in response to particular cues, including cellular stress and developmental signals (3). Interest in these structures has increased sharply as they have been recently linked to a number of human diseases, including neurodegenerative disorders like amyotrophic lateral sclerosis and spinocerebellar ataxia type II (4,5). Although the function of many of these granules is unknown, their conservation throughout evolution suggests that they serve an important biological role.Stress granules are one of the better-characterized instances of these cytoplasmic RNP complexes. They form in response to various types of cellular stress and in quiescent or G 0 cells (3, 6). They are highly dynamic structures, continuously shuttling components between the cytosol and the granule during the stress and disassembling rapidly following its cessation (7). In mammalian cells, stress granule formation is initiated by the translation arrest that occurs upon the stress-triggered phosphorylation of translation initiation factor 2␣ (eIF2␣) (8). Polysomes disassemble, and 48S preinitiation complexes containing naked mRNA accumulate and are bound by mRNA-binding proteins that contain low-complexity domains (8). These domains...

The eukaryotic cytoplasm contains a variety of ribonucleoprotein (RNP) granules in addition to the better-understood membrane-bound organelles. These granules form in response to specific stress conditions and contain a number of signaling molecules important for the control of cell growth and survival. However, relatively little is known about the mechanisms responsible for, and the ultimate consequences of, this protein localization. Here, we show that the Hrr25/CK1d protein kinase is recruited to cytoplasmic processing bodies (P-bodies) in an evolutionarily conserved manner. This recruitment requires Hrr25 kinase activity and the Dcp2 decapping enzyme, a core constituent of these RNP granules. Interestingly, the data indicate that this localization sequesters active Hrr25 away from the remainder of the cytoplasm and thereby shields this enzyme from the degradation machinery during these periods of stress. Altogether, this work illustrates how the presence within an RNP granule can alter the ultimate fate of the localized protein.KEYWORDS ribonucleoprotein granules; processing bodies; protein kinase; protein stability; Dcp2 decapping enzyme; casein kinase 1 T HE eukaryotic cell is subdivided into distinct functional areas by the presence of a variety of organelles. The best understood of these are the membrane-bound structures, like the nucleus, endoplasmic reticulum, and mitochondria. These traditional compartments are relatively stable and essential for the proper compartmentalization of the different reactions occurring in the cytoplasm. However, the cell also contains a collection of nonmembraneous organelles that are more dynamic in nature and form in response to particular cellular and environmental stimuli. Perhaps the two best characterized of these are the centrosome and nucleolus, which act as a microtubule-organizing center and a subnuclear site of ribosome assembly, respectively (Greenan et al. 2010;Brangwynne 2011;Brangwynne et al. 2011). This latter class also includes a number of recently identified cytoplasmic ribonucleoprotein (RNP) granules like the processing body (P-body) and stress granule (Anderson and Kedersha 2009;Balagopal and Parker 2009;Thomas et al. 2011). These cytoplasmic structures have been conserved through evolution and have been linked to a variety of human diseases, including certain neurodegenerative disorders, cancers, and autoimmune conditions (Li et al. 2013;Anderson et al. 2015). Despite this importance to human health, relatively little is known about the manner in which these RNP granules influence biological processes in the cell.These cytoplasmic RNP granules typically assemble in response to environmental stress or particular developmental cues (Thomas et al. 2011). Granule formation occurs as a consequence of the regulated coalescence of specific sets of proteins and translationally repressed mRNAs at discrete sites in the cytoplasm (Anderson and Kedersha 2009;Balagopal and Parker 2009). The presence of these core proteins often depends upon specific RNA-...

Transcription of protein-coding genes in mammalian cells is coordinated with pre-mRNA processing as well as the assembly and nuclear export of mRNPs. Btf (BCLAF1) and TRAP150 (THRAP3) were previously reported to associate with in vitro spliced mRNPs and also as a part of the spliceosome, suggesting they are involved in pre-mRNA processing. Btf and TRAP150 are serine-arginine-rich (SR) proteins with significant sequence similarity, but the extent of their functional overlap is not yet clear. We show that both Btf and TRAP150 localize at a constitutively active β-tropomyosin (BTM) reporter minigene locus in mammalian cells. Both proteins also localize at a U2OS 2-6-3 reporter gene locus in a RNA polymerase II (RNAPII) transcription-dependent manner. While Btf and TRAP150 showed some overlap with reporter RNA and other pre-mRNA processing factors at transcription loci, they showed the most precise overlap with the exon junction complex (EJC) protein Magoh. Since EJC components have roles in nuclear export, we examined nuclear/cytoplasmic mRNA distribution after Btf or TRAP150 knockdown. Btf depletion caused an increase of β-tropomyosin minigene reporter transcripts in the cytoplasm as well as global increase of endogenous polyadenylated RNA in the cytoplasm, while TRAP150 depletion did not. We provide evidence that Btf has functions distinct from TRAP150 in regulating the subcellular distribution of mRNAs in human cells.