Ribonucleoprotein Complex Biogenesis Research Articles

RNA-Binding Proteomics Reveals MATR3 Interacting with lncRNA SNHG1 To Enhance Neuroblastoma Progression.

The interaction of long noncoding RNAs (lncRNAs) with one or more RNA-binding proteins (RBPs) is important to a plethora of cellular and physiological processes. The lncRNA SNHG1 was reported to be aberrantly expressed and associated with poor patient prognosis in several cancers including neuroblastoma. However, the interacting RBPs and biological functions associated with SNHG1 in neuroblastoma remain unknown. In this study, we identified 283, 31, and 164 SNHG1-interacting proteins in SK-N-BE(2)C, SK-N-DZ, and SK-N-AS neuroblastoma cells, respectively, using a RNA-protein pull-down assay coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS). Twenty-four SNHG1-interacting RBPs were identified in common from these three neuroblastoma cell lines. RBPs MATR3, YBX1, and HNRNPL have the binding sites for SNHG1 predicted by DeepBind motif analysis. Furthermore, the direct binding of MATR3 with SNHG1 was validated by Western blot and confirmed by RNA immunoprecipitation assay (RIP). Coexpression analysis revealed that the expression of SNHG1 is positively correlated with MATR3 ( P = 3.402 × 10-13). The high expression of MATR3 is associated with poor event-free survival ( P = 0.00711) and overall survival ( P = 0.00064). Biological functions such as ribonucleoprotein complex biogenesis, RNA processing, and RNA splicing are significantly enriched and in common between SNHG1 and MATR3. In conclusion, we identified MATR3 as binding to SNHG1 and the interaction might be involved in splicing events that enhance neuroblastoma progression.

Global gene expression in muscle from fasted/refed trout reveals up-regulation of genes promoting myofibre hypertrophy but not myofibre production

BackgroundCompensatory growth is a phase of rapid growth, greater than the growth rate of control animals, that occurs after a period of growth-stunting conditions. Fish show a capacity for compensatory growth after alleviation of dietary restriction, but the underlying cellular mechanisms are unknown. To learn more about the contribution of genes regulating hypertrophy (an increase in muscle fibre size) and hyperplasia (the generation of new muscle fibres) in the compensatory muscle growth response in fish, we used high-density microarray analysis to investigate the global gene expression in muscle of trout during a fasting-refeeding schedule and in muscle of control-fed trout displaying normal growth.ResultsThe compensatory muscle growth signature, as defined by genes up-regulated in muscles of refed trout compared with control-fed trout, showed enrichment in functional categories related to protein biosynthesis and maturation, such as RNA processing, ribonucleoprotein complex biogenesis, ribosome biogenesis, translation and protein folding. This signature was also enriched in chromatin-remodelling factors of the protein arginine N-methyl transferase family. Unexpectedly, functional categories related to cell division and DNA replication were not inferred from the molecular signature of compensatory muscle growth, and this signature contained virtually none of the genes previously reported to be up-regulated in hyperplastic growth zones of the late trout embryo myotome and to potentially be involved in production of new myofibres, notably genes encoding myogenic regulatory factors, transmembrane receptors essential for myoblast fusion or myofibrillar proteins predominant in nascent myofibres.ConclusionGenes promoting myofibre growth, but not myofibre formation, were up-regulated in muscles of refed trout compared with continually fed trout. This suggests that a compensatory muscle growth response, resulting from the stimulation of hypertrophy but not the stimulation of hyperplasia, occurs in trout after refeeding. The generation of a large set of genes up-regulated in muscle of refed trout may yield insights into the molecular and cellular mechanisms controlling skeletal muscle mass in teleost and serve as a useful list of potential molecular markers of muscle growth in fish.

Pharmacological eEF 2K activation promotes cell death and inhibits cancer progression

Activation of the elongation factor 2 kinase (eEF2K) leads to the phosphorylation and inhibition of the elongation factor eEF2, reducing mRNA translation rates. Emerging evidence indicates that the regulation of factors involved in protein synthesis may be critical for controlling diverse biological processes including cancer progression. Here we show that inhibitors of the HIV aspartyl protease (HIV-PIs), nelfinavir in particular, trigger a robust activation of eEF2K leading to the phosphorylation of eEF2. Beyond its anti-viral effects, nelfinavir has antitumoral activity and promotes cell death. We show that nelfinavir-resistant cells specifically evade eEF2 inhibition. Decreased cell viability induced by nelfinavir is impaired in cells lacking eEF2K. Moreover, nelfinavir-mediated anti-tumoral activity is severely compromised in eEF2K-deficient engrafted tumors in vivo Our findings imply that exacerbated activation of eEF2K is detrimental for tumor survival and describe a mechanism explaining the anti-tumoral properties of HIV-PIs.

Proteomic and Genomic Analyses of the Rvb1 and Rvb2 Interaction Network upon Deletion of R2TP Complex Components

The highly conserved yeast R2TP complex, consisting of Rvb1, Rvb2, Pih1, and Tah1, participates in diverse cellular processes ranging from assembly of protein complexes to apoptosis. Rvb1 and Rvb2 are closely related proteins belonging to the AAA+ superfamily and are essential for cell survival. Although Rvbs have been shown to be associated with various protein complexes including the Ino80 and Swr1chromatin remodeling complexes, we performed a systematic quantitative proteomic analysis of their associated proteins and identified two additional complexes that associate with Rvb1 and Rvb2: the chaperonin-containing T-complex and the 19S regulatory particle of the proteasome complex. We also analyzed Rvb1 and Rvb2 purified from yeast strains devoid of PIH1 and TAH1. These analyses revealed that both Rvb1 and Rvb2 still associated with Hsp90 and were highly enriched with RNA polymerase II complex components. Our analyses also revealed that both Rvb1 and Rvb2 were recruited to the Ino80 and Swr1 chromatin remodeling complexes even in the absence of Pih1 and Tah1 proteins. Using further biochemical analysis, we showed that Rvb1 and Rvb2 directly interacted with Hsp90 as well as with the RNA polymerase II complex. RNA-Seq analysis of the deletion strains compared with the wild-type strains revealed an up-regulation of ribosome biogenesis and ribonucleoprotein complex biogenesis genes, down-regulation of response to abiotic stimulus genes, and down-regulation of response to temperature stimulus genes. A Gene Ontology analysis of the 80 proteins whose protein associations were altered in the PIH1 or TAH1 deletion strains found ribonucleoprotein complex proteins to be the most enriched category. This suggests an important function of the R2TP complex in ribonucleoprotein complex biogenesis at both the proteomic and genomic levels. Finally, these results demonstrate that deletion network analyses can provide novel insights into cellular systems.

Protein substrates of the arginine methyltransferase Hmt1 identified by proteome arrays.

Arginine methylation on nonhistone proteins is associated with a number of cellular processes including RNA splicing, protein localization, and the formation of protein complexes. In this manuscript, Saccharomyces cerevisiae proteome arrays carrying 4228 proteins were used with an antimethylarginine antibody to first identify 88 putatively arginine-methylated proteins. By treating the arrays with recombinant arginine methyltransferase Hmt1, 42 proteins were found to be possible substrates of this enzyme. Analysis of the putative arginine-methylated proteins revealed that they were predominantly nuclear or nucleolar in localization, consistent with the localization of Hmt1. Many are involved in known methylarginine-associated functions, such as RNA processing and ribonucleoprotein complex biogenesis, yet others are of newer classes, namely RNA/DNA helicases and tRNA-associated proteins. Using ex vivo methylation and MS/MS, a set of 12 proteins (Brr1, Dia4, Hts1, Mpp10, Mrd1, Nug1, Prp43, Rpa43, Rrp43, Spp381, Utp4, and Npl3), including the RNA helicase Prp43 and tRNA ligases Dia4 and Hts1, were all validated as Hmt1 substrates. Interestingly, the majority of these also had human orthologs, or family members, that have been documented elsewhere to carry arginine methylation. These results confirm arginine methylation as a widespread modification and Hmt1 as the major arginine methyltransferase in the S. cerevisiae cell.

Splicing Aberrations in Congenital Myasthenic Syndromes

The fidelity of splicing is indispensable for assuring rapidly changing cellular processes and physiological integrity of the cells. The precise biogenesis of ribo nucleoprotein complexes (RNPs) is therefore essential, which is ensured by proper complementation of RNA and RNA-binding proteins in a specific cellular context. Genetic mutations disrupting cis-acting splicing elements or compromising catalytic functions of trans-acting RNA-binding protein scan impair the biogenesis of functional RNPs and provoke pathological consequences. Analyses of splicing aberrations in human diseases not only allow us to understand the underlying maladies in pathological conditions, but also pave the way to gain insight into splicing regulation under physiological conditions. Dissection of the patho mechanisms of splicing defects in neuromuscular junction (NMJ) disorders has disclosed unprecedented roles of RNA-binding proteins in the neuromuscular signal transmission at the NMJ. This review focuses on splicing defects in congenital myasthenia syndromes (CMS), hereditary disorders caused by germ line mutations in molecules expressed at the NMJ. Splicing mis-regulations of the NMJ molecules that have been uncovered in the course of analyses of splicing mutations causing CMS can be extrapolated to other diseases caused by germ line or somatic mutations.

Yeast Rvb1 and Rvb2 Proteins Oligomerize As a Conformationally Variable Dodecamer with Low Frequency

Rvb1 and Rvb2 are conserved AAA+ (ATPases associated with diverse cellular activities) proteins found at the core of large multicomponent complexes that play key roles in chromatin remodeling, integrity of the telomeres, ribonucleoprotein complex biogenesis and other essential cellular processes. These proteins contain an AAA+ domain for ATP binding and hydrolysis and an insertion domain proposed to bind DNA/RNA. Yeast Rvb1 and Rvb2 proteins oligomerize primarily as heterohexameric rings. The six AAA+ core domains form the body of the ring and the insertion domains protrude from one face of the ring. Conversely, human Rvbs form a mixture of hexamers and dodecamers made of two stacked hexamers interacting through the insertion domains. Human dodecamers adopt either a contracted or a stretched conformation. Here, we found that yeast Rvb1/Rvb2 complexes when assembled in vivo mainly form hexamers but they also assemble as dodecamers with a frequency lower than 10%. Yeast dodecamers adopt not only the stretched and contracted structures that have been described for human Rvb1/Rvb2 dodecamers but also intermediate conformations in between these two extreme states. The orientation of the insertion domains of Rvb1 and Rvb2 proteins in these conformers changes as the dodecamer transitions from the stretched structure to a more contracted structure. Finally, we observed that for the yeast proteins, oligomerization as a dodecamer inhibits the ATPase activity of the Rvb1/Rvb2 complex. These results indicate that although human and yeast Rvb1 and Rvb2 proteins share high degree of homology, there are significant differences in their oligomeric behavior and dynamics.

MiR-629-3p Level Significantly Predicts Prognosis in Glioblastoma Patients Treated with Temozolomide Chemotherapy

Background: Glioblastoma is the most aggressive and common primary tumor of the central nervous system. Resistance to temozolomide, the first-line chemotherapy for glioblastoma, always leads to treatment failure. Further understanding of the molecular mechanisms of temozolomide resistance in glioblastoma is desperately needed. The purpose of this study was to identify miRNAs correlated with temozolomide treatment in glioblastoma paitents. Methods: miRNA expression microarray data of 82 glioblastoma samples were obtained from the Chinese Glioma Genome Atlas database (http://www.cgga.org.cn). In order to identify miRNAs correlated with temozolomide reponse, the significance of the association between each miRNA expression and overall survival of temozolomide treated glioblastoma patients was analyzed by univariate Cox proportional hazard regression analysis. Gene expression profile associated with miR-629-3p was determined using whole genome mRNA expression microarray data. Subsequently, biological progresses related to the miR-629-3p associated gene expression profile were identified by gene ontology analysis Results: The miR629-3p level was significantly correlated with overall survival in patients who underwent temozolomide chemotherapy, while a higher miR629-3p level failed to improve overall survival in patients who had not received temozolomide treatment. Gene ontology analysis showed that miR-629-3p associated genes were involved in the biological processes including translation, translational elongation, and ribonucleoprotein complex biogenesis. Conclusion: High-level miR629-3p expression was associated with prolonged overall survival in patients who underwent temozolomide chemotherapy, indicating that miR-629-3p may be a predictive biomarker for temozolomide response, a guide for clinical treatment decisions, and a target for glioblastoma therapy.

Karyopherin Alpha2 Is Essential for rRNA Transcription and Protein Synthesis in Proliferative Keratinocytes

Karyopherin proteins mediate nucleocytoplasmic trafficking and are critical for protein and RNA subcellular localization. Recent studies suggest KPNA2 expression is induced in tumor cells and is strongly associated with prognosis, although the precise roles and mechanisms of KPNA2 overexpression in proliferative disorders have not been defined. We found that KPNA2 expression is induced in various proliferative disorders of the skin such as psoriasis, Bowen’s disease, actinic keratosis, squamous cell carcinoma, Paget’s disease, Merkel cell carcinoma, and mycosis fungoides. siRNA-mediated KPNA suppression revealed that KPNA2 is essential for significant suppression of HaCaT proliferation under starvation conditions. Ribosomal RNA transcription and protein synthesis were suppressed by starvation combined with knockdown of KPNA (including KPNA2) expression. KPNA2 localized to the nucleolus and interacted with proteins associated with mRNA processing, ribonucleoprotein complex biogenesis, chromatin modification, and transcription, as demonstrated by tandem affinity purification and mass spectrometry. KPNA2 may be an important promoter of ribosomal RNA and protein synthesis in tumor cells.

Increased susceptibility of spinal muscular atrophy fibroblasts to camptothecin-induced cell death

Spinal muscular atrophy (SMA) is a neuromuscular disease caused by deletions or mutations in the telomeric copy of the survival motor neuron ( SMN1) gene. Although the SMN protein has been implicated in the biogenesis of ribonucleoprotein complexes and RNA processing, it is not clear how these functions contribute to the pathogenesis of SMA. To gain a further understanding of SMN function, we have investigated its role in cell survival in skin fibroblasts derived from SMA patients and age-matched controls. SMA fibroblasts exposed to camptothecin, a specific inhibitor of DNA topoisomerase I, consistently showed cell death at a lower concentration than normal controls. Treatment with other cell death-inducing agents did not cause differences in survival of SMA fibroblasts as compared with control fibroblasts. Camptothecin treatment resulted in activation of caspase-3 with generation of the caspase-3 cleavage product, poly ADP-ribose polymerase (PARP). Depletion of SMN protein by RNA interference in control fibroblasts increased caspase-3 activity, whereas transfection of SMA fibroblasts with wild-type SMN decreased caspase-3 activity. Our data demonstrate that SMA fibroblasts are more prone to some, but not all, death-stimuli. Vulnerability to death-stimuli is associated with decreased levels of SMN protein and is mediated by activation of caspase-3.