Characterization and in vivo functional analysis of the Schizosaccharomyces pombe ICLN gene.

During the early steps of snRNP biogenesis, the survival motor neuron (SMN) complex acts together with the methylosome, an entity formed by the pICln protein, WD45, and the PRMT5 methyltransferase. To expand our understanding of the functional relationship between pICln and SMN in vivo, we performed a genetic analysis of an uncharacterized Schizosaccharomyces pombe pICln homolog. Although not essential, the S. pombe ICln (SpICln) protein is important for optimal yeast cell growth. The human ICLN gene complements the Δicln slow-growth phenotype, demonstrating that the identified SpICln sequence is the bona fide human homolog. Consistent with the role of human pICln inferred from in vitro experiments, we found that the SpICln protein is required for optimal production of the spliceosomal snRNPs and for efficient splicing in vivo. Genetic interaction approaches further demonstrate that modulation of ICln activity is unable to compensate for growth defects of SMN-deficient cells. Using a genome-wide approach and reverse transcription (RT)-PCR validation tests, we also show that splicing is differentially altered in Δicln cells. Our data are consistent with the notion that splice site selection and spliceosome kinetics are highly dependent on the concentration of core spliceosomal components.

Genome-wide identification of mRNAs associated with the protein SMN whose depletion decreases their axonal localization.

Spinal muscular atrophy is a neuromuscular disease resulting from mutations in the SMN1 gene, which encodes the survival motor neuron (SMN) protein. SMN is part of a large complex that is essential for the biogenesis of spliceosomal small nuclear RNPs. SMN also colocalizes with mRNAs in granules that are actively transported in neuronal processes, supporting the hypothesis that SMN is involved in axonal trafficking of mRNPs. Here, we have performed a genome-wide analysis of RNAs present in complexes containing the SMN protein and identified more than 200 mRNAs associated with SMN in differentiated NSC-34 motor neuron-like cells. Remarkably, ~30% are described to localize in axons of different neuron types. In situ hybridization and immuno-fluorescence experiments performed on several candidates indicate that these mRNAs colocalize with the SMN protein in neurites and axons of differentiated NSC-34 cells. Moreover, they localize in cell processes in an SMN-dependent manner. Thus, low SMN levels might result in localization deficiencies of mRNAs required for axonogenesis.

Impaired minor tri-snRNP assembly generates differential splicing defects of U12-type introns in lymphoblasts derived from a type I SMA patient.

The survival of motor neuron (SMN) protein is essential for cytoplasmic assembly of spliceosomal snRNPs. Although the normal proportion of endogenous snRNAs is unevenly altered in spinal muscular atrophy (SMA) tissues, the biogenesis of individual snRNPs is not dramatically affected in SMN-deficient cells. The SMN protein is also required for normal Cajal body (CB) formation, but the functional consequences of CB disruption upon SMN deficiency have not yet been analyzed at the level of macromolecular snRNPs assembly. Here, we show that the SMN protein is required for tri-snRNPs formation and that the level of the minor U4atac/U6atac/U5 tri-snRNPs is dramatically decreased in lymphoblasts derived from a patient suffering from a severe form of SMA. We found also that splicing of some, but not all, minor introns is inhibited in these cells, demonstrating links between SMN deficiency and differential alterations of splicing events mediated by the minor spliceosome. Our results suggest that SMA might result from the inefficient splicing of one or only a few pre-mRNAs carrying minor introns and coding for proteins required for motor neurons function and/or organization.

HSP90 and its R2TP/Prefoldin-like cochaperone are involved in the cytoplasmic assembly of RNA polymerase II.

RNA polymerases are key multisubunit cellular enzymes. Microscopy studies indicated that RNA polymerase I assembles near its promoter. However, the mechanism by which RNA polymerase II is assembled from its 12 subunits remains unclear. We show here that RNA polymerase II subunits Rpb1 and Rpb3 accumulate in the cytoplasm when assembly is prevented and that nuclear import of Rpb1 requires the presence of all subunits. Using MS-based quantitative proteomics, we characterized assembly intermediates. These included a cytoplasmic complex containing subunits Rpb1 and Rpb8 associated with the HSP90 cochaperone hSpagh (RPAP3) and the R2TP/Prefoldin-like complex. Remarkably, HSP90 activity stabilized incompletely assembled Rpb1 in the cytoplasm. Our data indicate that RNA polymerase II is built in the cytoplasm and reveal quality-control mechanisms that link HSP90 to the nuclear import of fully assembled enzymes. hSpagh also bound the free RPA194 subunit of RNA polymerase I, suggesting a general role in assembling RNA polymerases.

Specific splicing defects in S. pombe carrying a degron allele of the Survival of Motor Neuron gene.

Spinal muscular atrophy results from deletions or mutations in the survival of motor neuron (SMN1) gene. The SMN protein has an essential role in the biogenesis of spliceosomal snRNPs, but the link between a defect in this process and specific splicing inhibition of pre-mRNAs has not been established. In this study, we report the construction of a temperature-degron (td) allele of the Schizosaccharomyces pombe SMN protein and show that its depletion at 37 degrees C affects splicing and formation of U1, U2, U4 and U5 snRNPs, but not of U6 and U3 ribonucleoproteins. The function of the tdSMN allele in snRNP assembly is already perturbed at 25 degrees C, suggesting a deleterious effect of the tag at this temperature. Using a genome-wide approach, we report that introns react unequally to lower levels of snRNPs in tdSMN cells and that increasing the length of the polypyrimidine tract can improve the splicing efficiency of some, but not all, affected introns. Altogether, our results suggest that the defects observed in tdSMN fission yeast cells mimic splicing deficits observed in SMN-deficient metazoan cells.

Characterization of a short isoform of human Tgs1 hypermethylase associating with small nucleolar ribonucleoprotein core proteins and produced by limited proteolytic processing.

Tgs1 is the hypermethylase responsible for m(3)G cap formation of U small nuclear RNAs (U snRNAs) and small nucleolar RNAs (snoRNAs). In vertebrates, hypermethylation of snRNAs occurs in the cytoplasm, whereas this process takes place in the nucleus for snoRNAs. Accordingly, the hypermethylase is found in both compartments with a diffuse localization in the cytoplasm and a concentration in Cajal bodies in the nucleoplasm. In this study, we report that the Tgs1 hypermethylase exists as two species, a full-length cytoplasmic isoform and a shorter nuclear isoform of 65-70 kDa. The short isoform exhibits methyltransferase activity and associates with components of box C/D and H/ACA snoRNPs, pointing to a role of this isoform in hypermethylation of snoRNAs. We also show that production of the short Tgs1 isoform is inhibited by MG132, suggesting that it results from proteasomal limited processing of the full-length Tgs1 protein. Together, our results suggest that proteasome maturation constitutes a mechanism regulating Tgs1 function by generating Tgs1 species with different substrate specificities, subcellular localizations, and functions.

Differential regulation of estrogen receptor alpha turnover and transactivation by Mdm2 and stress-inducing agents.

In mammalian cells, the level of estrogen receptor alpha (ERalpha) is rapidly decreased upon estrogen treatment, and this regulation involves proteasome degradation. Using different approaches, we showed that the Mdm2 oncogenic ubiquitin-ligase directly interacts with ERalpha in a ternary complex with p53 and is involved in the regulation of ERalpha turnover (both in the absence or presence of estrogens). Several lines of evidence indicated that this effect of Mdm2 required its ubiquitin-ligase activity and involved the ubiquitin/proteasome pathway. Moreover, in MCF-7 human breast cancer cells, various p53-inducing agents (such as UV irradiation) or treatment with RITA (which inhibits the interaction of p53 with Mdm2) stabilized ERalpha and abolished its 17beta-estradiol-dependent turnover. Interestingly, our data indicated that ligand-dependent receptor turnover was not required for efficient transactivation. Altogether, our results indicate that the Mdm2 oncoprotein and stress-inducing agents complexly and differentially regulate ERalpha stability and transcriptional activity in human cancer cells.

Depletion of SMN by RNA interference in HeLa cells induces defects in Cajal body formation.

Neuronal degeneration in spinal muscular atrophy (SMA) is caused by reduced expression of the survival of motor neuron (SMN) protein. The SMN protein is ubiquitously expressed and is present both in the cytoplasm and in the nucleus where it localizes in Cajal bodies. The SMN complex plays an essential role for the biogenesis of spliceosomal U-snRNPs. In this article, we have used an RNA interference approach in order to analyse the effects of SMN depletion on snRNP assembly in HeLa cells. Although snRNP profiles are not perturbed in SMN-depleted cells, we found that SMN depletion gives rise to cytoplasmic accumulation of a GFP-SmB reporter protein. We also demonstrate that the SMN protein depletion induces defects in Cajal body formation with coilin being localized in multiple nuclear foci and in nucleolus instead of canonical Cajal bodies. Interestingly, the coilin containing foci do not contain snRNPs but appear to co-localize with U85 scaRNA. Because Cajal bodies represent the location in which snRNPs undergo 2'-O-methylation and pseudouridylation, our results raise the possibility that SMN depletion might give rise to a defect in the snRNA modification process.

Nuclear localization properties of a conserved protuberance in the Sm core complex.

The nuclear import signal of snRNPs is composed of two essential components, the m(3)G cap structure of the snRNA and the Sm core NLS carried by the Sm protein core complex. We have previously proposed that, in yeast, this last determinant is represented by a basic-rich protuberance formed by the C-terminal extensions of Sm proteins. In mammals, as well as in other organisms, this component has not yet been precisely defined. Using GFP-Sm fusion constructs and immunolocalization as well as biochemical experiments, we show here that the C-terminal domains of human SmD1 and SmD3 proteins possess nuclear localization properties. Deletions of these domains increase cytoplasmic fluorescence and cytoplasmic localization of GFP-Sm mutant fusion alleles. Our results are consistent with a model in which the Sm core NLS is evolutionarily conserved and composed of a basic-rich protuberance formed by C-terminal domains of different Sm subtypes.