572 Biochemie
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The macromolecular SMN complex facilitates the formation of Sm-class ribonucleoproteins involved in mRNA processing (UsnRNPs). While biochemical studies have revealed key activities of the SMN complex, its structural investigation is lagging behind. Here we report on the identification and structural determination of the SMN complex from the lower eukaryote Schizosaccharomyces pombe, consisting of SMN, Gemin2, 6, 7, 8 and Sm proteins. The core of the SMN complex is formed by several copies of SMN tethered through its C-terminal alpha-helices arranged with alternating polarity. This creates a central platform onto which Gemin8 binds and recruits Gemins 6 and 7. The N-terminal parts of the SMN molecules extrude via flexible linkers from the core and enable binding of Gemin2 and Sm proteins. Our data identify the SMN complex as a multivalent hub where Sm proteins are collected in its periphery to allow their joining with UsnRNA.
Background:
Retinitis pigmentosa (RP) is an inherited eye disease characterized by the progressive degeneration of rod photoreceptor cells. Mutations in pre-mRNA splicing factors including PRPF31 have been identified as cause for RP, raising the question how mutations in general factors lead to tissue specific defects.
Results:
We have recently shown that the zebrafish serves as an excellent model allowing the recapitulation of key events of RP. Here we use this model to investigate two pathogenic mutations in PRPF31, SP117 and AD5, causing the autosomal dominant form of RP. We show that SP117 leads to an unstable protein that is mislocalized to the rod cytoplasm. Importantly, its overexpression does not result in photoreceptor degeneration suggesting haploinsufficiency as the underlying cause in human RP patients carrying SP117. In contrast, overexpression of AD5 results in embryonic lethality, which can be rescued by wild-type Prpf31. Transgenic retina-specific expression of AD5 reveals that stable AD5 protein is initially localized in the nucleus but later found in the cytoplasm concurrent with progressing rod outer segment degeneration and apoptosis. Importantly, we show for the first time in vivo that retinal transcripts are wrongly spliced in adult transgenic retinas expressing AD5 and exhibiting increased apoptosis in rod photoreceptors.
Conclusion:
Our data suggest that distinct mutations in Prpf31 can lead to photoreceptor degeneration through different mechanisms, by haploinsufficiency or dominant-negative effects. Analyzing the AD5 effects in our animal model in vivo, our data imply that aberrant splicing of distinct retinal transcripts contributes to the observed retina defects.
The formation of macromolecular complexes within the crowded environment of cells often requires aid from assembly chaperones. PRMT5 and SMN complexes mediate this task for the assembly of the common core of pre-mRNA processing small nuclear ribonucleoprotein particles (snRNPs). Core formation is initiated by the PRMT5-complex subunit pICln, which pre-arranges the core proteins into spatial positions occupied in the assembled snRNP. The SMN complex then accepts these pICln-bound proteins and unites them with small nuclear RNA (snRNA). Here, we have analyzed how newly synthesized snRNP proteins are channeled into the assembly pathway to evade mis-assembly. We show that they initially remain bound to the ribosome near the polypeptide exit tunnel and dissociate upon association with pICln. Coincident with its release activity, pICln ensures the formation of cognate heterooligomers and their chaperoned guidance into the assembly pathway. Our study identifies the ribosomal quality control hub as a site where chaperone-mediated assembly of macromolecular complexes can be initiated.
The Epstein-Barr Virus (EBV) -encoded EBNA2 protein, which is essential for the in vitro transformation of B-lymphocytes, interferes with cellular processes by binding to proteins via conserved sequence motifs. Its Arginine-Glycine (RG) repeat element contains either symmetrically or asymmetrically di-methylated arginine residues (SDMA and ADMA, respectively). EBNA2 binds via its SDMA-modified RG-repeat to the survival motor neurons protein (SMN) and via the ADMA-RG-repeat to the NP9 protein of the human endogenous retrovirus K (HERV-K (HML-2) Type 1). The hypothesis of this work was that the methylated RG-repeat mimics an epitope shared with cellular proteins that is used for interaction with target structures. With monoclonal antibodies against the modified RG-repeat, we indeed identified cellular homologues that apparently have the same surface structure as methylated EBNA2. With the SDMA-specific antibodies, we precipitated the Sm protein D3 (SmD3) which, like EBNA2, binds via its SDMA-modified RG-repeat to SMN. With the ADMA-specific antibodies, we precipitated the heterogeneous ribonucleoprotein K (hnRNP K). Specific binding of the ADMA-antibody to hnRNP K was demonstrated using E. coli expressed/ADMA-methylated hnRNP K. In addition, we show that EBNA2 and hnRNP K form a complex in EBV-infected B-cells. Finally, hnRNP K, when co-expressed with EBNA2, strongly enhances viral latent membrane protein 2A (LMP2A) expression by an unknown mechanism as we did not detect a direct association of hnRNP K with DNA-bound EBNA2 in gel shift experiments. Our data support the notion that the methylated surface of EBNA2 mimics the surface structure of cellular proteins to interfere with or co-opt their functional properties.
Accumulated common variants in the broader fragile X gene family modulate autistic phenotypes
(2015)
Fragile X syndrome (FXS) is mostly caused by a CGG triplet expansion in the fragile X mental retardation 1 gene (FMR1). Up to 60% of affected males fulfill criteria for autism spectrum disorder (ASD), making FXS the most frequent monogenetic cause of syndromic ASD. It is unknown, however, whether normal variants (independent of mutations) in the fragile X gene family (FMR1, FXR1, FXR2) and in FMR2 modulate autistic features. Here, we report an accumulation model of 8 SNPs in these genes, associated with autistic traits in a discovery sample of male patients with schizophrenia (N = 692) and three independent replicate samples: patients with schizophrenia (N = 626), patients with other psychiatric diagnoses (N = 111) and a general population sample (N = 2005). For first mechanistic insight, we contrasted microRNA expression in peripheral blood mononuclear cells of selected extreme group subjects with high-versus low-risk constellation regarding the accumulation model. Thereby, the brain-expressed miR-181 species emerged as potential "umbrella regulator", with several seed matches across the fragile X gene family and FMR2. To conclude, normal variation in these genes contributes to the continuum of autistic phenotypes.
Pre-mRNA splicing by the spliceosome is an essential step in the maturation of nearly all human mRNAs. Mutations in six spliceosomal proteins, PRPF3, PRPF4, PRPF6, PRPF8, PRPF31 and SNRNP200, cause retinitis pigmentosa (RP), a disease characterized by progressive photoreceptor degeneration. All splicing factors linked to RP are constituents of the U4/U6.U5 tri-snRNP subunit of the spliceosome, suggesting that the compromised function of this particle may lead to RP. Here, we report the identification of the p.R192H variant of the tri-snRNP factor PRPF4 in a patient with RP. The mutation affects a highly conserved arginine residue that is crucial for PRPF4 function. Introduction of a corresponding mutation into the zebrafish homolog of PRPF4 resulted in a complete loss of function in vivo. A series of biochemical experiments suggested that p.R192H disrupts the binding interface between PRPF4 and its interactor PRPF3. This interferes with the ability of PRPF4 to integrate into the tri-snRNP, as shown in a human cell line and in zebrafish embryos. These data suggest that the p.R192H variant of PRPF4 represents a functional null allele. The resulting haploinsufficiency of PRPF4 compromises the function of the tri-snRNP, reinforcing the notion that this spliceosomal particle is of crucial importance in the physiology of the retina.