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Late cytoplasmic maturation of the large ribosomal subunitBussiere, Cyril Luc Cassien 19 July 2012 (has links)
In all life ribosomes are the ribonucloprotein machines in charge of decoding the genetic code and synthesizing proteins. In eukaryotes, ribosomes are pre-assembled in the nucleus and exported to the cytoplasm where the final maturation steps occur prior to their partaking in translation. My dissertation work focused on aspects of the last two known steps of the pre-60S subunit cytoplasmic maturation. In the penultimate step, the anti-association factor Tif6 is released from 60S by the concerted action of the translocase-like GTPase Efl1 and Sdo1. The release of Tif6 is necessary for the ultimate maturation step, which involves release of the export adaptor Nmd3 by the ribosomal protein Rpl10 and the putative GTPase Lsg1.
Nmd3 is an essential export adaptor of the 60S subunit. Nmd3 binds to the ribosome in the nucleolus and is the last known trans-acting factor to be released from the subunit in the cytoplasm. In order to gain a better understanding of the molecular events leading to the release of Nmd3 from the 60S subunit I set out to identify the binding site of Nmd3 on 60S. In a collaboration with Dr Joachim Frank’s laboratory, we obtained a cryo-EM model of Nmd3 in a complex with 60S showing Nmd3 binding to the subunit joining face of the ribosome. This work provided the first visualization of an export factor on a ribosomal subunit.
The release of the anti-association factor Tif6 requires the translocase-like GTPase Efl1. Mutations in a loop of Rpl10 which embraces the P site tRNA trapped Tif6 on the subunit. These Rpl10 mutants could be suppressed by Tif6 mutants which have weakened affinity for the subunit. Mutations in Efl1 which suppress these Rpl10 mutants were also obtained. These suppressing mutations in Efl1 mapped to regions on the translocases eEF2 and EF-G important for conformational changes during translation. These results highlight molecular signaling between the P site, involving a loop of Rpl10, and Tif6, 90Å away. I propose that Efl1 promotes a translocation-like event during biogenesis of the 60S subunit prior to its first round of bona fide translation. / text
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Pancreatic Endocrine Tumourigenesis : Genes of potential importanceJohansson, Térèse A. January 2008 (has links)
<p>Understanding signalling pathways that control pancreatic endocrine tumour (PET) development and proliferation may reveal novel targets for therapeutic intervention. The pathogenesis for sporadic and hereditary PETs, apart from mutations of the <i>MEN1</i> and <i>VHL</i> tumour suppressor genes, is still elusive. The protein product of the <i>MEN1</i> gene, menin, regulates many genes. The aim of this thesis was to identify genes involved in pancreatic endocrine tumourigenesis, with special reference to Notch signalling.</p><p>Messenger RNA and protein expression of NOTCH1, HES1, HEY1, ASCL1, NEUROG3, NEUROD1, DLK1, POU3F4, PDX1, RPL10, DKK1 and TPH1 were studied in human PETs, sporadic and MEN 1, as well as in tumours from heterozygous <i>Men1</i> mice. For comparison, normal and <i>MEN1</i> non-tumourous human and mouse pancreatic specimens were used. Nuclear expression of HES1 was consistently absent in PETs. In mouse tumours this coincided with loss of menin expression, and there was a correlation between <i>Men1</i> expression and several Notch signalling factors. A new phenotype consisting of numerous menin-expressing endocrine cell clusters, smaller than islets, was found in <i>Men1</i> mice. Expression of NEUROG3 and NEUROD1 was predominantly localised to the cytoplasm in PETs and islets from MEN 1 patients and <i>Men1</i> mice, whereas expression was solely nuclear in wt mice. Differences in expression levels of Pou3f4, Rpl10 and Dlk1 between islets of <i>Men1</i> and wt mice were observed.</p><p>In addition, combined RNA interference and microarray expression analysis in the pancreatic endocrine cell line BON1 identified 158 target genes of ASCL1. For two of these, DKK1 (a negative regulator of the WNT/β-catenin signalling pathway) and TPH1, immunohistochemistry was performed on PETs. In concordance with the microarray finding, DKK1 expression showed an inverse relation to ASCL1 expression.</p><p>Altered subcellular localisation of HES1, NEUROD1 and NEUROG3 and down-regulation of DKK1 may contribute to tumourigenesis.</p>
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Pancreatic Endocrine Tumourigenesis : Genes of potential importanceJohansson, Térèse A. January 2008 (has links)
Understanding signalling pathways that control pancreatic endocrine tumour (PET) development and proliferation may reveal novel targets for therapeutic intervention. The pathogenesis for sporadic and hereditary PETs, apart from mutations of the MEN1 and VHL tumour suppressor genes, is still elusive. The protein product of the MEN1 gene, menin, regulates many genes. The aim of this thesis was to identify genes involved in pancreatic endocrine tumourigenesis, with special reference to Notch signalling. Messenger RNA and protein expression of NOTCH1, HES1, HEY1, ASCL1, NEUROG3, NEUROD1, DLK1, POU3F4, PDX1, RPL10, DKK1 and TPH1 were studied in human PETs, sporadic and MEN 1, as well as in tumours from heterozygous Men1 mice. For comparison, normal and MEN1 non-tumourous human and mouse pancreatic specimens were used. Nuclear expression of HES1 was consistently absent in PETs. In mouse tumours this coincided with loss of menin expression, and there was a correlation between Men1 expression and several Notch signalling factors. A new phenotype consisting of numerous menin-expressing endocrine cell clusters, smaller than islets, was found in Men1 mice. Expression of NEUROG3 and NEUROD1 was predominantly localised to the cytoplasm in PETs and islets from MEN 1 patients and Men1 mice, whereas expression was solely nuclear in wt mice. Differences in expression levels of Pou3f4, Rpl10 and Dlk1 between islets of Men1 and wt mice were observed. In addition, combined RNA interference and microarray expression analysis in the pancreatic endocrine cell line BON1 identified 158 target genes of ASCL1. For two of these, DKK1 (a negative regulator of the WNT/β-catenin signalling pathway) and TPH1, immunohistochemistry was performed on PETs. In concordance with the microarray finding, DKK1 expression showed an inverse relation to ASCL1 expression. Altered subcellular localisation of HES1, NEUROD1 and NEUROG3 and down-regulation of DKK1 may contribute to tumourigenesis.
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