探討人類hnRNP Q/R同源蛋白HRPR-1對於線蟲發育的影響

Investigate the influence of human hnRNP Q/R homolog, HRPR-1, in C. elegans development

陳怡妏(Yi-Wen Chen)

指導教授 ： 詹世鵬

國立臺灣大學/醫學院/微生物學研究所/碩士(2021年)

https://doi.org/10.6342/NTU202103415

摘要

異質性核醣核酸家族是一群參與在許多核醣核酸生合成步驟中的核醣核酸結合蛋白。錯誤調控的異質性核醣核酸已經被證實和癌症及神經退化性疾病相關。其中異質性核醣核酸 Q (hnRNP Q)又被稱為SYNCRIP，而它的突變會參與在脊髓性肌肉萎縮症及急性骨髓性白血病的發生。除此之外，它也會調控微小核醣核酸(microRNA) let-7的成熟。let-7最早是被發現在線蟲中調控發育時間點的異時性基因(heterochronic gene)。它屬於非編碼核醣核酸(non-coding RNA)且在各物種間具有高度的保留性。在人類中，低表現的let-7和癌症的發生息息相關。而在線蟲中let-7 (n2853)的突變也會造成let-7表現量下降，並且會造成線蟲的外陰爆裂(vulva bursting)以及接縫細胞(seam cell)無法在成蟲時完成最終分化而持續分裂，這也類似於癌細胞的分裂。let-7會被許多不同的蛋白調控，像是LIN-28以及異質性核醣核酸。在先前的實驗中發現在人類細胞株中，LIN-28和hnRNP Q有交互作用且降低hnRNP Q的表現能夠使let-7的表現量提升。由於let-7和LIN-28在物種間都具有高度的保留性，而且hnRNP Q在線蟲中也有同源蛋白HRPR-1，因此我們實驗室以線蟲作為模式生物來探討HRPR-1可能參與let-7和LIN-28的調節機制。我們實驗室先前觀察到，利用核醣核酸干擾(RNAi)的方式降低線蟲HRPR-1表現量可以抑制let-7(n2853)突變所造成的接縫細胞意外分裂的情形。因此我們想要觀察降低線蟲HRPR-1表現量是否可以抑制其他let-7(n2853)突變所造成的表現，並且探討HRPR-1在異時性路徑中所扮演的角色。
在本篇研究中我們觀察到降低線蟲HRPR-1表現可以抑制let-7(n2853)突變的線蟲外陰爆裂的現象進而轉變為外陰突出的現象，然而接縫細胞意外分裂的情形並不會受到影響。取而代之的是我們發現降低線蟲HRPR-1表現會使線蟲在第四幼蟲期(L4)發育到成蟲的過程發生遲滯，並且會使線蟲的性腺發育受到抑制。除此之外我們觀察到HRPR-1的降低並不會影響到let-7在異時性路徑的下游蛋白LIN-29。我們也發現在線蟲中HRPR-1並不會直接和LIN-28有交互作用，而這也顯示LIN-28和hnRNP Q的交互作用可能沒有在物種間被保留。然而有趣的是我們發現基於HRPR-1在胚胎及第一期幼蟲(L1)的表現量特別高，他可能是一個異時性基因且他表現的時間比LIN-28早一個階段。然而他在異時性路徑中所扮演的角色仍然是未知的且需要更多的研究來探討。

關鍵字

異質性核醣核酸蛋白 ； SYNCRIP ； let-7 微小核醣核酸 ； LIN-28 ； HRPR-1

並列摘要

Heterogeneous nuclear protein family is a large group of RNA binding proteins (RNPs) which involves in several steps in RNA processing. Dysregulation of hnRNPs could involve in cancers and neurodegeneration diseases. hnRNP Q is also known as SYNCRIP and the mutation of hnRNP Q has reported to participate in SMA and AML. Moreover, it also regulates the maturation of let-7 miRNA. let-7 was first identified as a heterochronic gene in C. elegans for development timing. It is a non-coding RNA and is highly evolutionarily conserved across different species. The decrease of let-7 is usually related to cancers. In C. elegans, the let-7(n2853) mutation causes reduction of let-7 expression, and induces vulva bursting and extra seam cells division at the young adult stage, which is similar to the cancer development in human. let-7 is regulated by several proteins such as LIN-28 and hnRNPs. Our lab previously found that human Lin28 interacts with hnRNP Q, and the reduction of hnRNP Q would increase let-7 expression. Since let-7 and LIN-28 are evolutionarily conserved, we sought to determine whether hnRNP Q homolog, HRPR-1 in C. elegans also plays a role in let-7 and LIN-28-related regulation. Previous results indicate that hrpr-1 RNAi can rescue the let-7(n2853) phenotype in seam cell proliferation and terminal differentiation. We want to investigate whether knocking down hrpr-1 can rescue other let-7(n2853) phenotypes, as well as the putative role of HRPR-1 in the heterochronic pathway.
In this study, we reveal that knockdown HRPR-1 rescues the vulva bursting of let-7 mutant worms but causes vulva protruding. However, the abnormal seam cell lineage of let-7(n2853) worms is not rescued by knocking down HRPR-1. Instead, the hrpr-1 RNAi causes the retardation of worm growth from L4 to adult and the gonad development arrest. Moreover, we reveal that knockdown HRPR-1 does not affect the expression of LIN-29, which is an indirect downstream of let-7 in the heterochronic pathway. Moreover, we could not detect interaction between C. elegans HRPR-1 and LIN-28 by co-immunoprecipitation. Thus, C. elegans HRPR-1 may not be involved in let-7 and LIN-28-related regulation. However, interestingly, we found that HRPR-1 behaves as a heterochronic gene, highly expressed in embryos and L1 worms, which is a stage earlier than LIN-28, then lower at the stages afterward. The role of HRPR-1 in heterochronic pathway is still unknown and need further investigation.

並列關鍵字

Heterogeneous nuclear protein ； SYNCRIP ； let-7 miRNA ； LIN-28 ； HRPR-1

參考文獻

Geuens, T., D. Bouhy, and V. Timmerman, The hnRNP family: insights into their role in health and disease. Hum Genet, 2016. 135(8): p. 851-67.

Dreyfuss, G., et al., hnRNP proteins and the biogenesis of mRNA. Annu Rev Biochem, 1993. 62: p. 289-321.

Krecic, A.M. and M.S. Swanson, hnRNP complexes: composition, structure, and function. Curr Opin Cell Biol, 1999. 11(3): p. 363-71.

Dreyfuss, G., L. Philipson, and I.W. Mattaj, Ribonucleoprotein particles in cellular processes. J Cell Biol, 1988. 106(5): p. 1419-25.

Gorlach, M., et al., Interaction of the RNA-binding domain of the hnRNP C proteins with RNA. EMBO J, 1992. 11(9): p. 3289-95.