Gemin5 proteolysis reveals a novel motif to identify L protease targets.

Translation of picornavirus RNA is governed by the internal ribosome entry site (IRES) element, directing the synthesis of a single polyprotein. Processing of the polyprotein is performed by viral proteases that also recognize as substrates host factors. Among these substrates are translation initiation factors and RNA-binding proteins whose cleavage is responsible for inactivation of cellular gene expression. Foot-and-mouth disease virus (FMDV) encodes two proteases, L(pro) and 3C(pro). Widespread definition of L(pro) targets suffers from the lack of a sufficient number of characterized substrates. Here, we report the proteolysis of the IRES-binding protein Gemin5 in FMDV-infected cells, but not in cells infected by other picornaviruses. Proteolysis was specifically associated with expression of L(pro), yielding two stable products, p85 and p57. In silico search of putative L targets within Gemin5 identified two sequences whose potential recognition was in agreement with proteolysis products observed in infected cells. Mutational analysis revealed a novel L(pro) target sequence that included the RKAR motif. Confirming this result, the Fas-ligand Daxx, was proteolysed in FMDV-infected and L(pro)-expressing cells. This protein carries a RRLR motif whose substitution to EELR abrogated L(pro) recognition. Thus, the sequence (R)(R/K)(L/A)(R) defines a novel motif to identify putative targets of L(pro) in host factors.

A novel role for Gemin5 in mRNA translation.

In eukaryotic cells translation initiation occurs through two alternative mechanisms, a cap-dependent operating in the majority of mRNAs, and a 5'-end-independent driven by internal ribosome entry site (IRES) elements, specific for a subset of mRNAs. IRES elements recruit the translation machinery to an internal position in the mRNA through a mechanism involving the IRES structure and several trans-acting factors. Here, we identified Gemin5 protein bound to the foot-and-mouth disease virus (FMDV) and hepatitis C virus (HCV) IRES using two independent approaches, riboproteomic analysis and immunoprecipitation of photocrosslinked factors. Functional analysis performed in Gemin5 shRNA-depleted cells, or in in vitro translation reactions, revealed an unanticipated role of Gemin5 in translation control as a down-regulator of cap-dependent and IRES-driven translation initiation. Consistent with this, pull-down assays showed that Gemin5 forms part of two distinct complexes, a specific IRES-ribonucleoprotein complex and an IRES-independent protein complex containing eIF4E. Thus, beyond its role in snRNPs biogenesis, Gemin5 also functions as a modulator of translation activity.

[The use of non-typical materials as a source of DNA in post-mortem diagnosis of spinal muscular atrophy]. / Zastosowanie nietypowego zródla DNA w posmiertnej diagnostyce radzeniowego zaniku miesni.

BACKGROUND AND PURPOSE: Patients affected with SMA I usually die in early childhood before the end of the second year of life. Clinical diagnosis is often doubtful--without any molecular verification--and isolated DNA is not available. In such cases predicting the outcome of consecutive pregnancies is not possible. It appears, however, that the families often keep some relics of the diseased child, such as milk teeth, and pieces of umbilical cord; a dried drop of blood used for the Guthrie test in newborn screening may be available. The aim of this study was the post mortem molecular diagnosis of SMA based on samples of DNA isolated from such remnants. MATERIAL AND METHODS: PCR technique was applied to amplify exons 7 and 8 of SMN gene; DraI and DdeI restriction enzymes were used to distinguish SMN1/SMN2 genes. RESULTS: The deletion of the SMN gene was found using DNA isolated from (1) a blot of dried blood, (2) a milk tooth and (3) dried umbilical cord of children who died several years ago without the molecular verification of the suspected SMA. In one case the analysis of DNA obtained from umbilical cord did not confirm the diagnosis of SMA. CONCLUSIONS: Our results showed that biological materials such as those mentioned above may be kept for a long period of time without degradation of DNA, which is still satisfactory for molecular diagnosis of SMA. This is very important for genetic counseling and offering a prenatal test to the families concerned.

SMN gene analysis of the spinal form of Charcot-Marie-Tooth disease.

The spinal form of Charcot-Marie-Tooth disease (spinal CMT) is a rare genetic disorder of the peripheral nervous system, the genetic basis of which remains unknown. To test the hypothesis that a defect of survival motor neuron (SMN), the determining gene for spinal muscular atrophy (SMA), would result in spinal CMT, 18 unrelated spinal CMT patients were studied. Nine of them were sporadic cases and the other nine belonged to unrelated autosomal dominant pedigrees. None of the 18 patients showed deletions involving SMN exons 7 or 8, the most frequent gene alteration found in SMA. In addition, haplotype analysis in two large autosomal dominant pedigrees showed that the 5q13 locus was not segregating with the spinal CMT locus. Therefore, neither the sporadic nor the familial cases of spinal CMT are associated with a SMN gene deletion, nor are the familial cases linked to the 5q13 region, indicating that this neuropathy is genetically different from SMA.