ABSTRACT
RNA helicases constitute a large protein family implicated in cellular RNA homeostasis and disease development. Here, we show that the RNA helicase IGHMBP2, linked to the neuromuscular disorder spinal muscular atrophy with respiratory distress type 1 (SMARD1), associates with polysomes and impacts translation of mRNAs containing short, GC-rich, and structured 5' UTRs. The absence of IGHMBP2 causes ribosome stalling at the start codon of target mRNAs, leading to reduced translation efficiency. The main mRNA targets of IGHMBP2-mediated regulation encode for components of the THO complex (THOC), linking IGHMBP2 to mRNA production and nuclear export. Accordingly, failure of IGHMBP2 regulation of THOC causes perturbations of the transcriptome and its encoded proteome, and ablation of THOC subunits phenocopies these changes. Thus, IGHMBP2 is an upstream regulator of THOC. Of note, IGHMBP2-dependent regulation of THOC is also observed in astrocytes derived from patients with SMARD1 disease, suggesting that deregulated mRNA metabolism contributes to SMARD1 etiology and may enable alternative therapeutic avenues.
Subject(s)
Muscular Atrophy, Spinal , Respiratory Distress Syndrome, Newborn , Humans , RNA, Messenger/genetics , Muscular Atrophy, Spinal/genetics , 5' Untranslated Regions , Homeostasis , DNA-Binding Proteins/genetics , Transcription Factors/geneticsABSTRACT
Spliceosomal snRNPs are multicomponent particles that undergo a complex maturation pathway. Human Sm-class snRNAs are generated as 3'-end extended precursors, which are exported to the cytoplasm and assembled together with Sm proteins into core RNPs by the SMN complex. Here, we provide evidence that these pre-snRNA substrates contain compact, evolutionarily conserved secondary structures that overlap with the Sm binding site. These structural motifs in pre-snRNAs are predicted to interfere with Sm core assembly. We model structural rearrangements that lead to an open pre-snRNA conformation compatible with Sm protein interaction. The predicted rearrangement pathway is conserved in Metazoa and requires an external factor that initiates snRNA remodeling. We show that the essential helicase Gemin3, which is a component of the SMN complex, is crucial for snRNA structural rearrangements during snRNP maturation. The SMN complex thus facilitates ATP-driven structural changes in snRNAs that expose the Sm site and enable Sm protein binding.
Subject(s)
RNA Precursors , RNA, Small Nuclear , Humans , RNA, Small Nuclear/metabolism , SMN Complex Proteins/metabolism , RNA Precursors/metabolism , HeLa Cells , Ribonucleoproteins, Small Nuclear/metabolism , snRNP Core Proteins/geneticsABSTRACT
Specialized assembly factors facilitate the formation of many macromolecular complexes in vivo. The formation of Sm core structures of spliceosomal U-rich small nuclear ribonucleoprotein particles (UsnRNPs) requires assembly factors united in protein arginine methyltransferase 5 (PRMT5) and survival motor neuron (SMN) complexes. We demonstrate that perturbations of this assembly machinery trigger complex cellular responses that prevent aggregation of unassembled Sm proteins. Inactivation of the SMN complex results in the initial tailback of Sm proteins on the PRMT5 complex, followed by down-regulation of their encoding mRNAs. In contrast, reduction of pICln, a PRMT5 complex subunit, leads to the retention of newly synthesized Sm proteins on ribosomes and their subsequent lysosomal degradation. Overexpression of Sm proteins under these conditions results in a surplus of Sm proteins over pICln, promoting their aggregation. Our studies identify an elaborate safeguarding system that prevents individual Sm proteins from aggregating, contributing to cellular UsnRNP homeostasis.
Subject(s)
Lysosomes/metabolism , RNA, Messenger/metabolism , Ribonucleoproteins, Small Nuclear/metabolism , Spliceosomes/metabolism , Autophagy , Down-Regulation , HeLa Cells , Humans , Ion Channels/genetics , Ion Channels/metabolism , Molecular Chaperones/genetics , Molecular Chaperones/metabolism , Phosphorylation , Protein Aggregates , Protein Stability , Protein-Arginine N-Methyltransferases/genetics , Protein-Arginine N-Methyltransferases/metabolism , Proteolysis , RNA Interference , RNA Stability , RNA, Messenger/genetics , Ribonucleoproteins, Small Nuclear/genetics , SMN Complex Proteins/genetics , SMN Complex Proteins/metabolism , Spliceosomes/genetics , Time Factors , TransfectionABSTRACT
Wolbachia surface protein (WSP), which is the most abundantly expressed protein of Wolbachia from the human filarial parasite Brugia malayi, was chosen for the present study. B-cell epitope prediction of the WSP protein sequence indicates a high antigenicity, surface probability and hydrophilicity by DNA STAR software analysis. ProPred analysis suggests the presence of HLA class II binding regions in the WSP protein that contribute to T-cell responses and isotype reactivity. In order to validate these findings, the gene coding for endosymbiont WSP was PCR-amplified from the genomic DNA of the human filarial parasite Brugia malayi and cloned in T-7 expression vector pRSET-A. Western blot and ELISA at the total IgG level with recombiant WSP indicated a significantly elevated reactivity in CP compared to MF, EN and NEN individuals. Isotype ELISA also suggested an elevated reactivity in CP patients at the IgG1 level. In contrast, WSP-specific IgG4 levels were found to be elevated in MF patients compared to CP and EN. Besides this, WSP-specific IgE levels indicated an elevated reactivity in CP and MF patients compared to normals. Observations from ELISA supported the in silico predictions that indicate the presence of B- and T-cell epitopes. Hence, a combinatorial approach of in silico predictions and wet-lab studies provides interesting insights into the role of Wolbachia proteins in filarial pathogenesis.
