線蟲HRP-2及其人類同源蛋白hnRNP Q/R在微小核醣核酸功能中扮演的角色

Abstract

微小核醣核酸(miRNAs)為一小段非編碼的核醣核酸，可和目標訊息核糖核酸的3端不轉譯區結合，進而抑制目標基因的轉譯或是使其被降解。異質核醣核酸蛋白(Heterogeneous nuclear ribonucleoproteins , hnRNPs)為一核醣核酸結合蛋白的家族之一，參與許多核醣核酸的代謝路徑，其中包含參與miRNA的生合成以及功能當中，例如人類hnRNP A1被發現能夠抑制let-7 miRNA的生合成。從線蟲到人類中let-7以及let-7對於lin-41的調控具有保守性，在本篇論文中，我們降低了在線蟲中已知的hnRNP來觀察這些hnRNP對於let-7的調控是否有影響。在線蟲幼蟲晚期階段，let-7會降低LIN-41的表現，間接活化被LIN-41抑制的成蟲專屬轉錄因子LIN-29，進而促進細胞終端分化並且進入成蟲細胞階段。let-7的功能缺失造成線蟲生殖孔不正常發育、皮下接縫細胞(hypodermal seam cells)終端分化異常以及LIN-29所活化的膠原蛋白基因col-19表現量降低。在let-7亞效等位基因（hypomorphic allele）線蟲突變種let-7(n2853)中降低HRP-2能夠抑制這些let-7異時基因性狀(heterochronic phenotypes)，但並不能抑制lin-29無效等位基因(null allele)線蟲突變種lin-29(n333)的性狀缺失。HRP-2的減少並不影響let-7的表現量。這些觀察顯示HRP-2參與在lin-29的上游來影響let-7對於目標基因的調控，並且不會影響let-7的生合成。此外，我們發現降低HRP-2也能抑制let-60/Ras 功能增強所導致的多生殖孔性狀，let-60/Ras被let-7旁系同源(paralogous)miRNA miR-84調控，進而決定生殖孔前驅細胞的分化命運，然而，我們的結果顯示HRP-2對於生殖孔的調控是miR-84-independent的。HRP-2除了先前研究發現參與在選擇性剪接的功能之外，我們的研究也發現HRP-2能夠參與miRNA的功能進而調控基因的表現，至少參與了let-7的功能。但在人類Huh7細胞中降低線蟲HRP-2的人類同源蛋白hnRNP Q，對於let-7的表現量以及let-7對TRIM71/LIN41的調控沒有影響。有趣的是，在細胞中降低另一個HRP-2的同源蛋白hnRNP R能夠顯著的降低TRIM71/LIN41的表現量，但可能是透過3端不轉譯區以外未知的機制來調控TRIM71/LIN41。總結以上結果，我們推測HRP-2能調控let-7在線蟲異時基因let-7-lin-41-lin-29調控路徑中的功能，但在人類Huh7細胞中HRP-2同源蛋白hnRNP Q/R卻不影響具保守性的let-7對TRIM71/LIN41調控關係。

MicroRNAs (miRNAs) are small non-coding RNAs that regulate genes by binding to the 3′ untranslated region (3′ UTR) of target mRNAs and trigger translational repression and/or mRNA degradation. Heterogeneous nuclear ribonucleoproteins (hnRNPs) are a family of RNA-binding proteins involved in many RNA metabolisms, including miRNA biogenesis and functions. For example, human hnRNP A1 has been implicated in inhibiting biogenesis of the let-7 miRNA. let-7 and the regulation of lin-41 by let-7 are conserved from nematodes to humans. In this study, we knocked down several known C. elegans hnRNPs by RNAi and examined the effects on let-7-mediated regulation. In C. elegans, let-7 down-regulates LIN-41 in the late larval stages and indirectly activates the adult-specific transcription factor LIN-29 that is suppressed by LIN-41, and hence promotes cell terminal differentiation and adult cell fates. Dysfunction of let-7 results in abnormal vulva development, attenuated terminal differentiation of hypodermal seam cells and low expression of col-19, an adult collagen gene directly activated by LIN-29. We found that knockdown of hrp-2 suppressed the let-7 heterochronic phenotypes in animals carrying a hypomorphic let-7(n2853) allele, but not those with a null lin-29(n333) allele. Depletion of HRP-2 did not change let-7 levels. These observations suggest that HRP-2 plays a role in let-7-mediated gene regulation in the heterochronic gene pathway, likely upstream of lin-29, without affecting let-7 biogenesis. On the other hand, hrp-2 knockdown suppressed the multiple vulva (Muv) phenotype caused by gain-of-function mutations of let-60/Ras, a direct target of the let-7 paralogous miRNA miR-84 that determines vulval precursor cell differentiation. However, we found that the suppression is miR-84-independent. Nevertheless, our findings suggest that HRP-2, in addition to its previously reported function in alternative splicing, controls gene expression via miRNA-mediated regulation, at least for let-7. In human Huh7 cells, depletion of hnRNP Q, the homolog of C. elegans HRP-2, did not affect let-7 levels or the regulation of TRIM71/LIN41 by let-7. Interestingly, knockdown of the other HRP-2 homolog, hnRNP R, significantly reduced TRIM71/LIN41 expression, however, by an unknown mechanism outside regulation through the 3’ UTR. Taken together, we propose that HRP-2 controls let-7-mediated regulation in the let-7-lin-41-lin-29 axis in the C. elegans heterochronic gene pathway, while its homologous proteins hnRNP Q/R in human Huh7 cells may not affect the conserved let-7-TRIM71/LIN41 regulation.