ß 2014. Published by The Company of Biologists Ltd | Journal of Cell Science (2014) 127, 812–827 doi:10.1242/jcs.137703 RESEARCH ARTICLE Time-resolved quantitative proteomics implicates the core snRNP protein SmB together with SMN in neural trafficking Alan R. Prescott1, Alexandra Bales2, John James1, Laura Trinkle-Mulcahy3 and Judith E. Sleeman2,* ABSTRACT defects in SMA, which affect predominantly spinal motor neurons, are caused by a decreased capacity for splicing snRNP The biogenesis of splicing snRNPs (small nuclear ribonucleoproteins) maturation or by defects in other, as yet uncharacterized, roles for is a complex process, beginning and ending in the nucleus of the cell SMN in neural cells. Although numerous reports have been made but including key stages that take place in the cytoplasm. In particular, of accumulations of Sm proteins and/or SMN in the cytoplasm, the SMN (survival motor neuron) protein complex is required for primarily in cells overexpressing SMN or mutant SMN (Liu and addition of the core Sm proteins to the snRNP. Insufficiency of SMN Gall, 2007; Pellizzoni et al., 1998; Sleeman et al., 2003), the results in the inherited neurodegenerative condition, spinal muscular cellular location and organization of the addition of Sm proteins atrophy (SMA). Details of the physical organization of the cytoplasmic to the snRNA during assembly of the snRNP is unknown. stages of snRNP biogenesis are unknown. Here, we use time- Furthermore, the identity and role of SMN-rich cytoplasmic resolved quantitative proteomics to identify proteins that associate accumulations has been a subject of debate. In Drosophila,U preferentially with either newly assembled or mature splicing bodies, which contain snRNAs and SMN (Liu and Gall, 2007), snRNPs. We identified highly mobile SmB protein-trafficking have been proposed to play a role in snRNP assembly, whereas vesicles in neural cells, which are dependent on the cellular levels SMN-rich structures in mammalian neural cells, reported in some of SMN and SmB for their morphology and mobility. We propose that cases to lack Sm proteins, have been implicated in the these represent a family of related vesicles, some of which play a role localization of mRNAs (Fallini et al., 2010; Fallini et al., 2011; in snRNP biogenesis and some that might play more diverse roles in Fan and Simard, 2002; Hubers et al., 2011; Jablonka et al., 2001; cellular RNA metabolism. Peter et al., 2011; Sharma et al., 2005; Todd et al., 2010a; Todd et al., 2010b; Zhang et al., 2006), with a proposed role in the local KEY WORDS: SILAC protemics, snRNPs, Spinal muscular atrophy, translation of mRNAs required for motor neuron function. Vesicles, SMN, Survival of motor neuron protein Following the assembly of the Sm protein core, the partially assembled snRNP is imported into the nucleus. Once imported, snRNPs accumulate transiently in the Cajal body (CB) where the INTRODUCTION core snRNA is modified. Further maturation of snRNPs involves The five uridine-rich spliceosomal snRNPs, U1, U2, U4, U5 and the addition of a number of additional proteins specific to each U6 are essential components of the spliceosome required for class of snRNP (for example, U1 snRNP contains the proteins removal by splicing of introns from pre-mRNA. With the U1A, U170K and U1C). Again, it is not known exactly where or exception of U6 snRNP, splicing U snRNPs have a complex how these stages are carried out, but it is assumed that these pathway of biogenesis involving cytoplasmic and nuclear stages proteins are added after the snRNP has been re-imported into the (reviewed by Fischer et al., 2011). Following their transcription nucleus. Mature snRNPs are believed to be stored in splicing by RNA polymerase II in the nucleus, the small nuclear RNAs speckles from which they are recruited to active spliceosomes (snRNAs) are exported into the cytoplasm where early events in when required (reviewed by Spector and Lamond, 2011). snRNP maturation occur including hypermethylation of the 59 In order to dissect the molecular mechanisms and subcellular cap of the snRNA and trimming of the 39 end. Additionally, a organization of different stages of splicing snRNP biogenesis, in heptameric ring of core Sm proteins is assembled onto the Sm- particular cytoplasmic stages of potential relevance to SMA, we binding site of the snRNA. Assembly of the Sm core onto the used a time-resolved quantitative proteomic approach. Differential snRNA requires the SMN (survival motor neurons) complex labeling of cells containing FP–tagged newly assembled snRNPs (reviewed by Battle et al., 2006). This process has been the (transiently transfected with FP–SmB) versus FP-tagged mature subject of considerable attention because lowered levels of SMN snRNPs (lines stably expressing FP–SmB) was achieved by result in the inherited neurodegenerative condition spinal culturing cells in tissue culture media containing different muscular atrophy (SMA) (Lefebvre et al., 1995; Lefebvre et al., isotopes of the essential amino acids arginine and lysine. This 1997). It is currently unclear whether the specific pathological technique, known as stable isotope labeling by amino acids in culture (SILAC) (Ong et al., 2002), was then combined with affinity purification and mass spectrometry to facilitate comparison 1 2 College of Life Sciences, University of Dundee, Dundee DD1 5EH, UK. School of the interactomes of snRNPs at these two distinct stages. of Biology, University of St Andrews, BMS, North Haugh, St Andrews KY16 9ST, UK. 3Department of Cellular and Molecular Medicine and Ottawa Institute of Investigations informed by these data revealed a family of novel Systems Biology, University of Ottawa, 451 Smyth Road, Ottawa, ON K1H 8M5, cytoplasmic vesicles, enriched in SmB and SMN. Unexpectedly, Canada. these vesicles appear to be heterogeneous in their constituents other *Author for correspondence ([email protected]) than SMN and SmB, suggesting that they are involved in several aspects of cellular RNA metabolism. The SmB- and SMN-rich Received 30 June 2013; Accepted 30 November 2013 vesicles are upregulated during neural differentiation, disrupted by Journal of Cell Science 812 RESEARCH ARTICLE Journal of Cell Science (2014) 127, 812–827 doi:10.1242/jcs.137703 overexpression of SMN or reduced expression of SmB and are highly Direct comparison of the degree of enrichment of detected mobile in neurites, implicating them in the pathology of SMA. proteins above background with mature (log H:L) versus newly assembled (log M:L) snRNPs clearly identifies interaction RESULTS partners, both known and novel, and highlights key differences Expression of the core snRNP protein SmB enables the preferential between the two pools (Fig. 2A). For example, although Sm labeling of mature and newly made snRNPs in a time-dependent and proteins (yellow) and several other snRNP proteins (orange) are concentration-independent manner clearly enriched in both samples, non-snRNP splicing factors Our previous analysis of the splicing snRNP maturation pathway (green) are more enriched with mature snRNPs, whereas a larger using FP-tagged Sm proteins revealed that newly expressed Sm number of transport factors (purple) co-purify with newly proteins are first detected diffusely in the cytoplasm, before assembled snRNPs. The log H:M ratios directly measure accumulating transiently in Cajal bodies prior to forming the relative enrichment with mature (H) versus newly assembled steady-state localization of mature splicing snRNPs in splicing (M) snRNPs, and information about relative abundance can be speckles (Sleeman and Lamond, 1999). Because the Sm proteins added by plotting these ratios versus the summed intensities of all are assembled into snRNPs before reaching speckles (Sleeman peptides identified for that particular protein (normalized by et al., 2001; Sleeman 2007) (supplementary material Fig. S1), this molecular weight) (Fig. 2B). As expected, the most highly defines a pathway of maturation for splicing snRNPs. This enriched protein was the YFP–SmB bait protein. Although pathway does not influence the localization of pre-existing slightly more was pulled down from transiently transfected cells snRNPs within the cell. HeLa cells constitutively expressing (newly assembled snRNPs), it still fell below the chosen YFP–SmB can be fused with cells constitutively expressing CFP– threshold (.1 log above or below the median log H:M) and SmB, and the import into the nucleus of newly assembled was therefore not significant. The most highly enriched and snRNPs monitored. This results in differently localized mature abundant mature snRNP interactors were the Sm and other and newly assembled snRNPs (Fig. 1A). In cells stably expres- snRNP proteins. Importantly, a large number of non-snRNP sing FP–SmB, the majority of FP-tagged snRNPs are mature, splicing factors are also present in the Sm protein interactome, with only a few newly assembled snRNPs present. Importantly demonstrating conclusively for the first time that FP-tagged Sm for our experimental approach, the use of fluorescently tagged Sm proteins are not only assembled into splicing snRNPs, but are also proteins enables the maturity of tagged snRNPs to be assessed recruited into the spliceosome. Most of the proteins known to be using live cell microscopy immediately before processing the involved in snRNP maturation including the SMN complex and cells for proteomic analysis. the PRMT5 complex involved in methylation of Sm proteins are plotted near or slightly below