利用生物資訊方法偵測程序性核糖體移碼之研究

Abstract

程序性核糖體移碼是一種重新編碼的機制, 藉由這個機制核糖體會在某個特定位置上, 從原本的零讀碼框切換到−1 或+1 的讀碼框(其中切換到−1 的讀碼框最為常見), 然後在新的讀碼框中繼續蛋白質的轉譯。此機制會導致另一個蛋白質的表現, 而這個蛋白質與未發生 程序性核糖體移碼時所產生的蛋白質不同。時至今日, 許多生物體, 包含病毒、細菌和真核生物等, 已被發現利用程序性核糖體移碼機制來增加其基因表現的多樣性, 或是進行其基因的調控。此外, 也有文獻指出, 對利用此機制的病毒, 即便僅僅改變很小的移碼效率就可抑制其繁殖。這意味著程序性核糖體移碼位置的發現與辨識, 在為抗病毒藥劑找尋新標的的研究上, 可能扮演著重要的角色。在這篇論文中, 我們利用序列模式辨認的方法, 輔之以結構與功能生物資訊學, 設計出一套更有效的演算法, 可以偵測出基因序列中會發生程序性核糖體移碼的位置。根據這個演算法, 我們也已實作出一個名為PRooF (Programmed Ribosomal Frameshifting 的簡稱) 的網路伺服器, 可供生物學家做線上的分析。PRooF 的正確性也已經過了許多含有一或二個程序性核糖體移碼的基因序列的測試, 而且測試結果也與用現存 最新的工具所得的結果做了比較。比較結果指出PRooF 在偵測的敏感度上的確有著大幅度的改進。特別的是, PRooF 所偵測出在移碼位置後方的RNA 二級結構大多數為H-type pseudoknots 和bulged helixes。相較於simple stem-loops, 以上兩種結構被廣泛認為更能 促使核糖體移碼的發生。

Programmed ribosomal frameshifting (PRF) is a recoding mechanism by which the translational ribosome switches from the initial (zero) reading frame to one of the two alternative reading frame (either mostly −1 or +1) at a specific position and continues its translation in the new frame. As a result, the recoding of PRF leads to an expression of an alternative protein, which is different from that produced by standard translation. To date, many organisms, including viruses, bacteria and eukaryotes, have been found to utilize the PRF mechanism for increasing the diversity of gene expression or for gene regulation. In addition, it has been reported that, for viruses that use PRF, even small alterations in their frameshifting efficiencies can inhibit viral propagation, suggesting that the PRF discovery and identification may play a crucial role in identifying new targets for antiviral agents. In this thesis, using a pattern recognition approach aided by structural and functional bioinformatics, we have designed a more effective algorithm to detect −1 and +1 PRF sites in a genomic sequence. This algorithm has also been implemented as a web server, called PRooF (short for Programmed Ribosomal Frameshifting), for online analysis. The accuracy of PRooF was tested with several genomic sequences, each of which has already been known to carry one or two PRF sites, by comparing its testing results with those obtained by the latest existing program. Consequently, the experimental results show that PRooF indeed greatly improves detection sensitivity. In particular, most of the predicted stimulatory RNA structures downstream of slippery sites are H-type pseudoknots and bulged helixes, both of which are widely believed to promote the ribosomal frameshifting events more efficiently than simple stem-loops.