TOR signaling regulates liquid phase separation of the SMN complex governing snRNP biogenesis.

The activity of the SMN complex in promoting the assembly of pre-mRNA processing UsnRNPs correlates with condensation of the complex in nuclear Cajal bodies. While mechanistic details of its activity have been elucidated, the molecular basis for condensation remains unclear. High SMN complex phosphorylation suggests extensive regulation. Here, we report on systematic siRNA-based screening for modulators of the capacity of SMN to condense in Cajal bodies and identify mTOR and ribosomal protein S6 kinase ß-1 as key regulators. Proteomic analysis reveals TOR-dependent phosphorylations in SMN complex subunits. Using stably expressed or optogenetically controlled phospho mutants, we demonstrate that serine 49 and 63 phosphorylation of human SMN controls the capacity of the complex to condense in Cajal bodies via liquid-liquid phase separation. Our findings link SMN complex condensation and UsnRNP biogenesis to cellular energy levels and suggest modulation of TOR signaling as a rational concept for therapy of the SMN-linked neuromuscular disorder spinal muscular atrophy.

Stakeholders' perspectives on biobank-based genomic research: systematic review of the literature.

The success of biobank-based genomic research is widely dependent on people's willingness to donate their tissue. Thus, stakeholders' opinions should be considered in the development of best practice guidelines for research and recruiting participants. We systematically analyzed the empirical literature describing different stakeholders' views towards ethical questions with regard to type of consent, data sharing and return of incidental findings. Patients are more open to one-time general consent than the public. Only a small proportion desires recontact if the research aim changed. A broad consent model would prevent only a small proportion of patients from participating in research. Although professionals are concerned about a risk of reidentification, patients and the public support data sharing and find that the benefit of research outweighs the potential risk of reidentification. However, they desire detailed information about the privacy protection measures. Regarding the return of incidental findings, the public and professionals focus on clinically actionable results, whereas patients are interested in receiving as much information as possible. For professionals, concrete guidelines that help managing the return of incidental findings should be warranted. For this it would be helpful addressing the different categories - actionable, untreatable and inheritable diseases - upfront with patients and public.

The catalytically inactive tyrosine phosphatase HD-PTP/PTPN23 is a novel regulator of SMN complex localization.

The survival motor neuron (SMN) complex fulfils essential functions in the assembly of snRNPs, which are key components in the splicing of pre-mRNAs. Little is known about the regulation of SMN complex activity by posttranslational modification despite its complicated phosphorylation pattern. Several phosphatases had been implicated in the regulation of SMN, including the nuclear phosphatases PPM1G and PP1γ. Here we systematically screened all human phosphatase gene products for a regulatory role in the SMN complex. We used the accumulation of SMN in Cajal bodies of intact proliferating cells, which actively assemble snRNPs, as a readout for unperturbed SMN complex function. Knockdown of 29 protein phosphatases interfered with SMN accumulation in Cajal bodies, suggesting impaired SMN complex function, among those the catalytically inactive, non-receptor-type tyrosine phosphatase PTPN23/HD-PTP. Knockdown of PTPN23 also led to changes in the phosphorylation pattern of SMN without affecting the assembly of the SMN complex. We further show interaction between SMN and PTPN23 and document that PTPN23, like SMN, shuttles between nucleus and cytoplasm. Our data provide the first comprehensive screen for SMN complex regulators and establish a novel regulatory function of PTPN23 in maintaining a highly phosphorylated state of SMN, which is important for its proper function in snRNP assembly.

Phosphoregulation of the human SMN complex.

The survival motor neuron (SMN) complex is a macromolecular machine comprising 9 core proteins: SMN, Gemins2-8 and unrip in vertebrates. It performs tasks in RNA metabolism including the cytoplasmic assembly of spliceosomal small nuclear ribonucleoprotein particles (snRNPs). The SMN complex also localizes to the nucleus, where it accumulates in Cajal Bodies (CB) and may function in transcription and/or pre-mRNA splicing. The SMN complex is subject to extensive phosphorylation. Detailed understanding of SMN complex regulation necessitates a comprehensive analysis of these post-translational modifications. Here, we report on the first comprehensive phosphoproteome analysis of the intact human SMN complex, which identify 48 serine/threonine phosphosites in the complex. We find that 7 out of 9 SMN components of the intact complex are phosphoproteins and confidently place 29 phosphorylation sites, 12 of them in SMN itself. By the generation of multi non-phosphorylatable or phosphomimetic variants of SMN, respectively, we address to which extent phosphorylation regulates SMN complex function and localization. Both phosphomimetic and non-phosphorylatable variants assemble into intact SMN complexes and can compensate the loss of endogenous SMN in snRNP assembly at least to some extent. However, they partially or completely fail to target to nuclear Cajal bodies. Moreover, using a mutant of SMN, which cannot be phosphorylated on previously reported tyrosine residues, we provide first evidence that this PTM regulates SMN localization and nuclear accumulation. Our data suggest complex regulatory cues mediated by phosphorylation of serine/threonine and tyrosine residues, which control the subcellular localization of the SMN complex and its accumulation in nuclear CB.

The function of spliceosome components in open mitosis.

Spatial separation of eukaryotic cells into the nuclear and cytoplasmic compartment permits uncoupling of DNA transcription from translation of mRNAs and allows cells to modify newly transcribed pre mRNAs extensively. Intronic sequences (introns), which interrupt the coding elements (exons), are excised ("spliced") from pre-mRNAs in the nucleus to yield mature mRNAs. This not only enables alternative splicing as an important source of proteome diversity, but splicing is also an essential process in all eukaryotes and knock-out or knock-down of splicing factors frequently results in defective cell proliferation and cell division. However, higher eukaryotes progress through cell division only after breakdown of the nucleus ("open mitosis"). Open mitosis suppresses basic nuclear functions such as transcription and splicing, but allows separate, mitotic functions of nuclear proteins in cell division. Mitotic defects arising after loss-of-function of splicing proteins therefore could be an indirect consequence of compromised splicing in the closed nucleus of the preceding interphase or reflect a direct contribution of splicing proteins to open mitosis. Although experiments to directly distinguish between these two alternatives have not been reported, indirect evidence exists for either hypotheses. In this review, we survey published data supporting an indirect function of splicing in open mitosis or arguing for a direct function of spliceosomal proteins in cell division.

Cajal-body formation correlates with differential coilin phosphorylation in primary and transformed cell lines.

Cajal bodies (CBs) are nuclear structures that are thought to have diverse functions, including small nuclear ribonucleoprotein (snRNP) biogenesis. The phosphorylation status of coilin, the CB marker protein, might impact CB formation. We hypothesize that primary cells, which lack CBs, contain different phosphoisoforms of coilin compared with that found in transformed cells, which have CBs. Localization, self-association and fluorescence recovery after photobleaching (FRAP) studies on coilin phosphomutants all suggest this modification impacts the function of coilin and may thus contribute towards CB formation. Two-dimensional gel electrophoresis demonstrates that coilin is hyperphosphorylated in primary cells compared with transformed cells. mRNA levels of the nuclear phosphatase PPM1G are significantly reduced in primary cells and expression of PPM1G in primary cells induces CBs. Additionally, PPM1G can dephosphorylate coilin in vitro. Surprisingly, however, expression of green fluorescent protein alone is sufficient to form CBs in primary cells. Taken together, our data support a model whereby coilin is the target of an uncharacterized signal transduction cascade that responds to the increased transcription and snRNP demands found in transformed cells.