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

Piñeiro D, Ramajo J, Bradrick SS, Martínez-Salas E - Nucleic Acids Res. (2012)

Bottom Line: Proteolysis was specifically associated with expression of L(pro), yielding two stable products, p85 and p57.Confirming this result, the Fas-ligand Daxx, was proteolysed in FMDV-infected and L(pro)-expressing cells.Thus, the sequence (R)(R/K)(L/A)(R) defines a novel motif to identify putative targets of L(pro) in host factors.

View Article: PubMed Central - PubMed

Affiliation: Centro de Biología Molecular Severo Ochoa, Consejo Superior de Investigaciones Cientificas-Universidad Autonoma de Madrid, Cantoblanco, 28049 Madrid, Spain.

ABSTRACT
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.

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Gemin5/p85 represses translation in a cell-free system. (A) Equal amounts of the bicistronic pBIC RNA, encoding CAT as the first cistron and luciferase as the IRES-dependent cistron, were added to RRL previously incubated during 15 min with increasing amounts of Gemin5/845–1508 RNA. Samples were analysed by SDS–PAGE 12% followed by autoradiography. (B) The intensity of luciferase and CAT proteins observed in four independent assays, normalized to the value obtained with the pBIC RNA alone, is represented against the amount of Gemin5 RNA added to the translation assay.
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gks172-F6: Gemin5/p85 represses translation in a cell-free system. (A) Equal amounts of the bicistronic pBIC RNA, encoding CAT as the first cistron and luciferase as the IRES-dependent cistron, were added to RRL previously incubated during 15 min with increasing amounts of Gemin5/845–1508 RNA. Samples were analysed by SDS–PAGE 12% followed by autoradiography. (B) The intensity of luciferase and CAT proteins observed in four independent assays, normalized to the value obtained with the pBIC RNA alone, is represented against the amount of Gemin5 RNA added to the translation assay.

Mentions: We have previously shown that Gemin5 binds directly to the IRES element and acts as a translation repressor using either cell free extracts or shRNA-depleted cells (20). Thus, it was of interest to determine whether the Gemin5 proteolysis products retained their capacity to repress translation. To this end, expression of the polypeptide 845–1508 was used to monitor the effect on IRES-dependent translation by measuring the efficiency of translation of bicistronic constructs bearing the FMDV IRES between CAT and luciferase reporter genes in rabbit reticulocyte lysates. The results indicated that the Gemin5/845–1508 amino acid product exerted a moderate dose-dependent repressor effect on IRES-driven translation of luciferase, as well as 5′ end-dependent translation of CAT (Figure 6A), although the repressor effect was somewhat more effective on the first cistron than that of IRES-dependent translation (Figure 6B), and in both cases smaller that that exerted by the uncleaved Gemin5 (p170) (20). Thus, we conclude that the p85 product only partially retains the translation repression capacity.Figure 6.


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

Piñeiro D, Ramajo J, Bradrick SS, Martínez-Salas E - Nucleic Acids Res. (2012)

Gemin5/p85 represses translation in a cell-free system. (A) Equal amounts of the bicistronic pBIC RNA, encoding CAT as the first cistron and luciferase as the IRES-dependent cistron, were added to RRL previously incubated during 15 min with increasing amounts of Gemin5/845–1508 RNA. Samples were analysed by SDS–PAGE 12% followed by autoradiography. (B) The intensity of luciferase and CAT proteins observed in four independent assays, normalized to the value obtained with the pBIC RNA alone, is represented against the amount of Gemin5 RNA added to the translation assay.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3367203&req=5

gks172-F6: Gemin5/p85 represses translation in a cell-free system. (A) Equal amounts of the bicistronic pBIC RNA, encoding CAT as the first cistron and luciferase as the IRES-dependent cistron, were added to RRL previously incubated during 15 min with increasing amounts of Gemin5/845–1508 RNA. Samples were analysed by SDS–PAGE 12% followed by autoradiography. (B) The intensity of luciferase and CAT proteins observed in four independent assays, normalized to the value obtained with the pBIC RNA alone, is represented against the amount of Gemin5 RNA added to the translation assay.
Mentions: We have previously shown that Gemin5 binds directly to the IRES element and acts as a translation repressor using either cell free extracts or shRNA-depleted cells (20). Thus, it was of interest to determine whether the Gemin5 proteolysis products retained their capacity to repress translation. To this end, expression of the polypeptide 845–1508 was used to monitor the effect on IRES-dependent translation by measuring the efficiency of translation of bicistronic constructs bearing the FMDV IRES between CAT and luciferase reporter genes in rabbit reticulocyte lysates. The results indicated that the Gemin5/845–1508 amino acid product exerted a moderate dose-dependent repressor effect on IRES-driven translation of luciferase, as well as 5′ end-dependent translation of CAT (Figure 6A), although the repressor effect was somewhat more effective on the first cistron than that of IRES-dependent translation (Figure 6B), and in both cases smaller that that exerted by the uncleaved Gemin5 (p170) (20). Thus, we conclude that the p85 product only partially retains the translation repression capacity.Figure 6.

Bottom Line: Proteolysis was specifically associated with expression of L(pro), yielding two stable products, p85 and p57.Confirming this result, the Fas-ligand Daxx, was proteolysed in FMDV-infected and L(pro)-expressing cells.Thus, the sequence (R)(R/K)(L/A)(R) defines a novel motif to identify putative targets of L(pro) in host factors.

View Article: PubMed Central - PubMed

Affiliation: Centro de Biología Molecular Severo Ochoa, Consejo Superior de Investigaciones Cientificas-Universidad Autonoma de Madrid, Cantoblanco, 28049 Madrid, Spain.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus