The Impact of p70S6 Kinase-Dependent Phosphorylation of Gemin2 in UsnRNP Biogenesis.

The survival motor neuron (SMN) complex is a multi-megadalton complex involved in post-transcriptional gene expression in eukaryotes via promotion of the biogenesis of uridine-rich small nuclear ribonucleoproteins (UsnRNPs). The functional center of the complex is formed from the SMN/Gemin2 subunit. By binding the pentameric ring made up of the Sm proteins SmD1/D2/E/F/G and allowing for their transfer to a uridine-rich short nuclear RNA (UsnRNA), the Gemin2 protein in particular is crucial for the selectivity of the Sm core assembly. It is well established that post-translational modifications control UsnRNP biogenesis. In our work presented here, we emphasize the crucial role of Gemin2, showing that the phospho-status of Gemin2 influences the capacity of the SMN complex to condense in Cajal bodies (CBs) in vivo. Additionally, we define Gemin2 as a novel and particular binding partner and phosphorylation substrate of the mTOR pathway kinase ribosomal protein S6 kinase beta-1 (p70S6K). Experiments using size exclusion chromatography further demonstrated that the Gemin2 protein functions as a connecting element between the 6S complex and the SMN complex. As a result, p70S6K knockdown lowered the number of CBs, which in turn inhibited in vivo UsnRNP synthesis. In summary, these findings reveal a unique regulatory mechanism of UsnRNP biogenesis.

Comprehensive Comparison of the Capacity of Functionalized Sepharose, Magnetic Core, and Polystyrene Nanoparticles to Immuno-Precipitate Procalcitonin from Human Material for the Subsequent Quantification by LC-MS/MS.

Sepsis is a life-threatening organ dysfunction caused by a dysregulated host response to infection. The fast and accurate diagnosis of sepsis by procalcitonin (PCT) has emerged as an essential tool in clinical medicine. Although in use in the clinical laboratory for a long time, PCT quantification has not yet been standardized. The International Federation of Clinical Chemistry working group on the standardization of PCT (IFCC-WG PCT) aims to provide an LC-MS/MS-based reference method as well as the highest metrological order reference material to address this diagnostic need. Here, we present the systematic evaluation of the efficiency of an immuno-enrichment method, based on functionalized Sepharose, magnetic-core, or polystyrene (latex) nano-particles, to quantitatively precipitate PCT from different human sample materials. This method may be utilized for both mass spectrometric and proteomic purposes. In summary, only magnetic-core nano-particles functionalized by polyclonal PCT antibodies can fulfil the necessary requirements of the international standardization of PCT. An optimized method proved significant benefits in quantitative and specific precipitation as well as in the subsequent LC-MS/MS detection of PCT in human serum samples or HeLa cell extract. Based on this finding, further attempts of the PCT standardization process will utilize a magnetic core-derived immuno-enrichment step, combined with subsequent quantitative LC-MS/MS detection.

Phosphorylation of pICln by the autophagy activating kinase ULK1 regulates snRNP biogenesis and splice activity of the cell.

The spliceosome, responsible for all mature protein-coding transcripts of eukaryotic intron-containing genes, consists of small uridine-rich nuclear ribonucleoproteins (UsnRNPs). The assembly of UsnRNPs depends, on one hand, on the arginine methylation of Sm proteins catalyzed by the PRMT5 complex. On the other hand, it depends on the phosphorylation of the PRMT5 subunit pICln by the Uncoordinated Like Kinase 1 (ULK1). In consequence, phosphorylation of pICln affects the stability of the UsnRNP assembly intermediate, the so-called 6 S complex. The detailed mechanisms of phosphorylation-dependent integrity and subsequent UsnRNP assembly of the 6 S complex in vivo have not yet been analyzed. By using a phospho-specific antibody against ULK1-dependent phosphorylation sites of pICln, we visualize the intracellular distribution of phosphorylated pICln. Furthermore, we detect the colocaliphosphor-pICln1 with phospho-pICln by size-exclusion chromatography and immunofluorescence techniques. We also show that phosphorylated pICln is predominantly present in the 6 S complex. The addition of ULK1 to in vitro produced 6 S complex, as well as the reconstitution of ULK1 in ULK1-deficient cells, increases the efficiency of snRNP biogenesis. Accordingly, inhibition of ULK1 and the associated decreased pICln phosphorylation lead to accumulation of the 6 S complex and reduction in the spliceosomal activity of the cell.

Development of Macrocyclic PRMT5-Adaptor Protein Interaction Inhibitors.

The PRMT5-MEP50 methyltransferase is a major target for anticancer drug discovery, and modulators of its interactions with different regulatory proteins are in high demand because they modulate PRMT5 substrate selectivity. We describe a strategy for the development of a PRMT5/adaptor protein PPI inhibitor, which includes the design and synthesis of macrocyclic peptides based on the motif for the interaction of PRMT5 with its adaptor protein RioK1. After the initial exploration of different macrocycle sizes and cyclization linkages, analysis of a peptide library identified hot spots for the variation of the amino acid structure. The incorporation of nonproteinogenic amino acids into the macrocyclic peptide led to a potent cyclic PRMT5 binding peptide (Ki = 66 nM), which selectively inhibits the interaction of PRMT5 with the adaptor proteins RioK1 and pICln (IC50 = 654 nM) but not with the alternative adaptor protein MEP50. The inhibitor is a promising tool for further biological investigation of this intriguing protein interface.

NF90/NFAR (nuclear factors associated with dsRNA) - a new methylation substrate of the PRMT5-WD45-RioK1 complex.

Protein-arginine methylation is a common posttranslational modification, crucial to various cellular processes, such as protein-protein interactions or binding to nucleic acids. The central enzyme of symmetric protein arginine methylation in mammals is the protein arginine methyltransferase 5 (PRMT5). While the methylation reaction itself is well understood, recruitment and differentiation among substrates remain less clear. One mechanism to regulate the diversity of PRMT5 substrate recognition is the mutual binding to the adaptor proteins pICln or RioK1. Here, we describe the specific interaction of Nuclear Factor 90 (NF90) with the PRMT5-WD45-RioK1 complex. We show for the first time that NF90 is symmetrically dimethylated by PRMT5 within the RG-rich region in its C-terminus. Since upregulation of PRMT5 is a hallmark of many cancer cells, the characterization of its dimethylation and modulation by specific commercial inhibitors in vivo presented here may contribute to a better understanding of PRMT5 function and its role in cancer.

An essential role of the autophagy activating kinase ULK1 in snRNP biogenesis.

The biogenesis of small uridine-rich nuclear ribonucleoproteins (UsnRNPs) depends on the methylation of Sm proteins catalyzed by the methylosome and the subsequent action of the SMN complex, which assembles the heptameric Sm protein ring onto small nuclear RNAs (snRNAs). In this sophisticated process, the methylosome subunit pICln (chloride conductance regulatory protein) is attributed to an exceptional key position as an 'assembly chaperone' by building up a stable precursor Sm protein ring structure. Here, we show that-apart from its autophagic role-the Ser/Thr kinase ULK1 (Uncoordinated [unc-51] Like Kinase 1) functions as a novel key regulator in UsnRNP biogenesis by phosphorylation of the C-terminus of pICln. As a consequence, phosphorylated pICln is no longer capable to hold up the precursor Sm ring structure. Consequently, inhibition of ULK1 results in a reduction of efficient UsnRNP core assembly. Thus ULK1, depending on its complex formation, exerts different functions in autophagy or snRNP biosynthesis.