To investigate the roles of C. elegans hnRNPs MSI-1 andHRP-2 in let-7-mediated lin-41 repression
碩士 === 國立臺灣大學 === 微生物學研究所 === 105 === Heterogeneous nuclear ribonucleoproteins (hnRNPs) have been demonstrated to participate in almost all RNA metabolisms, including transcription, splcing, RNA transport, translation and RNA degradation. In the past decade, hnRNPs were also found in regulation of b...
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ndltd-TW-105NTU053810152019-05-15T23:39:40Z http://ndltd.ncl.edu.tw/handle/y2v7uj To investigate the roles of C. elegans hnRNPs MSI-1 andHRP-2 in let-7-mediated lin-41 repression 研究線蟲中異質核醣核蛋白MSI-1以及HRP-2在let-7抑制lin-41的路徑中扮演的角色 Yu-Yong Su 蘇育揚 碩士 國立臺灣大學 微生物學研究所 105 Heterogeneous nuclear ribonucleoproteins (hnRNPs) have been demonstrated to participate in almost all RNA metabolisms, including transcription, splcing, RNA transport, translation and RNA degradation. In the past decade, hnRNPs were also found in regulation of biogenesis and/or function for certain microRNAs (miRNAs). miRNAs are a group of small noncoding regulatory RNAs that inhibit gene expression at the post-transcriptional level via binding to the 3′UTRs of target mRNAs and triggering translational repression and mRNA degradation. Among them, let-7, one of the founding members, regulates cell proliferation and terminal differentiation in Caenorhabditis elegans majorly by inhibiting the heterochronic gene lin-41 at the larva-to-adult transition of the nematode. The LIN-41 protein represses expression of the adult-specific transcription factor lin-29, hence loss-of-function mutations in lin-41 strongly exhibit precocious phentotypes and let-7 loss-of-function usually results in retardation due to the inability to inhibt lin-41. The let-7-mediated repression on lin-41 has been demonstarted to be evolutionarily conserved from the nematodes to humans and malfunction of let-7 frequently is associated with a variety of cancers. Here, we preform RNAi screen in the nematode C. elegans to seek hnRNPs that are required for biogenesis and/or function of the let-7 miRNA. We found that depletion of MSI-1 by RNAi suppressed the retarded phenotypes caused by the hypomorphic temperature-sensitive let-7(n2853) mutation, such as delayed terminal differentiation of seam cells and the defects in vulval development, but not the retardation of the lin-29(n333) null mutant. In addition, msi-1 knockdown seemed to induce precocious expression of lin-29, monitored by observation on a lin-29::mCherry reporter that should be expressed in seam cells initially from the L4 stage. Moreover, we utilized GFP reporters fused with the lin-41 3′UTR, lin-41 3′UTRΔLCS (LCS stands for let-7 complementary sequences) or the 3′UTR of unc-54, which is not regulated by let-7, to determine whether depletion of MSI-I indeed affects let-7 regulation on lin-41 and saw a positive relevance. Thus, we propose that MSI-1 acts as a negative regulatory factor for let-7 to repress lin-41 in the let-7-lin-41-lin-29 axis. Interestingly, our results in northern blotting assays showed no changes in the let-7 level upon msi-1 knockdown, suggesting that MSI-1 may acts on let-7 functioning but not biogenesis, perhaps recognizing target sequences in the lin-41 3′UTR or repressing translation of the lin-41 mRNA. Further investigation is needed to determine the molecular mechanism underlying. Shih-Peng Chan 詹世鵬 2017 學位論文 ; thesis 154 zh-TW |
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碩士 === 國立臺灣大學 === 微生物學研究所 === 105 === Heterogeneous nuclear ribonucleoproteins (hnRNPs) have been demonstrated to participate in almost all RNA metabolisms, including transcription, splcing, RNA transport, translation and RNA degradation. In the past decade, hnRNPs were also found in regulation of biogenesis and/or function for certain microRNAs (miRNAs). miRNAs are a group of small noncoding regulatory RNAs that inhibit gene expression at the post-transcriptional level via binding to the 3′UTRs of target mRNAs and triggering translational repression and mRNA degradation. Among them, let-7, one of the founding members, regulates cell proliferation and terminal differentiation in Caenorhabditis elegans majorly by inhibiting the heterochronic gene lin-41 at the larva-to-adult transition of the nematode. The LIN-41 protein represses expression of the adult-specific transcription factor lin-29, hence loss-of-function mutations in lin-41 strongly exhibit precocious phentotypes and let-7 loss-of-function usually results in retardation due to the inability to inhibt lin-41. The let-7-mediated repression on lin-41 has been demonstarted to be evolutionarily conserved from the nematodes to humans and malfunction of let-7 frequently is associated with a variety of cancers. Here, we preform RNAi screen in the nematode C. elegans to seek hnRNPs that are required for biogenesis and/or function of the let-7 miRNA. We found that depletion of MSI-1 by RNAi suppressed the retarded phenotypes caused by the hypomorphic temperature-sensitive let-7(n2853) mutation, such as delayed terminal differentiation of seam cells and the defects in vulval development, but not the retardation of the lin-29(n333) null mutant. In addition, msi-1 knockdown seemed to induce precocious expression of lin-29, monitored by observation on a lin-29::mCherry reporter that should be expressed in seam cells initially from the L4 stage. Moreover, we utilized GFP reporters fused with the lin-41 3′UTR, lin-41 3′UTRΔLCS (LCS stands for let-7 complementary sequences) or the 3′UTR of unc-54, which is not regulated by let-7, to determine whether depletion of MSI-I indeed affects let-7 regulation on lin-41 and saw a positive relevance. Thus, we propose that MSI-1 acts as a negative regulatory factor for let-7 to repress lin-41 in the let-7-lin-41-lin-29 axis. Interestingly, our results in northern blotting assays showed no changes in the let-7 level upon msi-1 knockdown, suggesting that MSI-1 may acts on let-7 functioning but not biogenesis, perhaps recognizing target sequences in the lin-41 3′UTR or repressing translation of the lin-41 mRNA. Further investigation is needed to determine the molecular mechanism underlying.
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author2 |
Shih-Peng Chan |
author_facet |
Shih-Peng Chan Yu-Yong Su 蘇育揚 |
author |
Yu-Yong Su 蘇育揚 |
spellingShingle |
Yu-Yong Su 蘇育揚 To investigate the roles of C. elegans hnRNPs MSI-1 andHRP-2 in let-7-mediated lin-41 repression |
author_sort |
Yu-Yong Su |
title |
To investigate the roles of C. elegans hnRNPs MSI-1 andHRP-2 in let-7-mediated lin-41 repression |
title_short |
To investigate the roles of C. elegans hnRNPs MSI-1 andHRP-2 in let-7-mediated lin-41 repression |
title_full |
To investigate the roles of C. elegans hnRNPs MSI-1 andHRP-2 in let-7-mediated lin-41 repression |
title_fullStr |
To investigate the roles of C. elegans hnRNPs MSI-1 andHRP-2 in let-7-mediated lin-41 repression |
title_full_unstemmed |
To investigate the roles of C. elegans hnRNPs MSI-1 andHRP-2 in let-7-mediated lin-41 repression |
title_sort |
to investigate the roles of c. elegans hnrnps msi-1 andhrp-2 in let-7-mediated lin-41 repression |
publishDate |
2017 |
url |
http://ndltd.ncl.edu.tw/handle/y2v7uj |
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