2008-11-13

[影片] 簡介「RNA干擾」現象之短片

lavande 譯



When long double-stranded RNAs were injected into a worms’ gonad, a standard way of introducing transgenes into worms, they blocked the expression of endogenous genes in a sequence-specific manner.

In eukaryotes, most protein-coding genes are transcribed by RNA polymerase II, which generates pre-mRNAs that are then processed to form mature mRNAs. These mRNAs are then transported from the nucleus to the cytoplasm, where they are translated. 

RNAi is a recently discovered process that can regulate endogenous gene expression. In plants, RNAi can be set in motion by genomically encoded short regulatory RNAs known as micro-RNAs. In algae, worms, and flies, RNAi can be activated by endogenous transposition. In plants and cultured insect cells, RNAi also has a role in anti-viral defense, in which viral double-stranded RNAs are targeted for destruction by the RNAi machinery.

When long double-stranded RNAs enter a cell, they are recognized and cleaved by Dicer, which is a member of the RNase III family of double-stranded RNA-specific endonucleases. Cleavage by Dicer creates short double-stranded RNAs that are characterized by two nucleotide long, 3’ overhangs. These are called small-interfering, or siRNAs. In worms, flies and mammals, siRNAs can form a ribonucleic protein complex called RISC, or RNAi silencing complex. This complex includes an unidentified nuclease that has been nicknamed Slicer.

A single-stranded siRNA that is coupled to RISC then binds to a target mRNA in a sequence-specific manner. This binding mediates target mRNA cleavage by Slicer. The site of cleavage falls in the middle of the region of siRNA complementarity. The cleaved mRNA can be recognized by the cell as being aberrant, and then destroyed.  This prevents translation from occurring, silencing the expression of the gene from which the mRNA was transcribed.

In plants, the aberrant RNA that result from RISC-mediated cleavage can also serve as a template for RNA-dependent RNA polymerase, or RdRp, to make a new double-stranded RNA molecule. This process relies on unprimed RNA synthesis, in which the aberrant RNA is used as a template. The resulting double-stranded RNA is a substrate for Dicer activity, which generates more siRNAs.

In some organisms with endogenous RNAi mechanisms, for example, fungi, plants, worms, and mammals, RNAi also involves another amplification step. In this step, single-stranded siRNAs not associated with RISC bind to their target mRNAs in a sequence-specific way, and serve as a primer for RdRp to polymerize the antisense RNA strand. Such specificity is intrinsically sensitive to natural sequence variation. The double-stranded RNA molecule that is created serves as a substrate for Dicer, which cuts it into siRNAs.

In turn, these siRNAs can either unwind and prime RNA-dependent RNA polymerization, or together with RISC mediates the cleavage of target mRNAs. This amplification, coupled with RNAi spreading between cells through an unknown mechanism, is thought to underlie germ line transmission of RNAi in worms.

「RNA干擾」現象首度於線蟲中發現。研究人員將長段的雙股RNA注射到線蟲生殖腺中(這是一種將基因引入線蟲常用的方式),發現這些RNA會阻撓線蟲內源性基因之表現,而受影響基因的序列恰與RNA序列互補。

在真核生物中,蛋白質的生成方式大概是這樣子的:第二型RNA聚合酶以蛋白質表達基因為模板,轉錄製造出pre-mRNA。pre-mRNA會進一步被修飾成成熟的mRNA,而後自細胞核內被運送出去,在細胞質中被轉譯製造出蛋白質。

近幾年才被發現的RNA干擾現象是生物調節體內基因表現的一種機制。植物基因組能製作出一種短小的調節性RNA(名為micro-RNA),用以啟動RNA干擾機制。在藻類、線蟲、果蠅中,RNA干擾現象也可能由基因轉位作用所活化。另外在植物和人工培養的昆蟲細胞中,干擾性RNA亦能破壞外來病毒的雙股RNA,發揮抗病毒之效用。

當長段雙股RNA進入細胞後,會被一種名為Dicer的雙股RNA內切酶(屬於第三類RNA水解酶)所辨識和切割。Dicer切割後會產生一類在3’端有2個核苷酸突出之小段雙股RNA,即siRNA,意為「小片段的干擾RNA」。在線蟲、果蠅和哺乳類中,siRNA能與其他蛋白質組合成為一核酸蛋白複合體,名為RISC,意為「能沉默基因之RNA干擾複合體」。此複合體中包含一個核酸內切酶,其身份尚未確知,研究人員暱稱它為Slicer。

接著,與RISC複合體連結在一起的單股siRNA會和序列互補之mRNA相互結合,Slicer進一步在兩者序列互補區段之中心點切割此mRNA。斷裂的mRNA會被細胞認定為異常物質而被摧毀,後續轉譯步驟無從進行,自然阻斷了mRNA上所載基因之表現,達到基因沉默之效。

在植物中,RISC切割後所產生的異常RNA還可作為RNA聚合酶合成RNA時的模板,在沒有引子的情況下製造出新的雙股RNA。這些雙股RNA又可被Dicer切割,進而產生更多siRNA。

而在藻類、植物、線蟲和哺乳類等體內具備RNA干擾機制的生物中,RNA干擾現象還可透過另一種方式擴大其效應。方法是這樣的:未與RISC複合體連結的單股siRNA與序列互補之mRNA結合後,可作為RNA聚合酶合成RNA時的引子,進而製造出反股RNA。(siRNA與mRNA的結合具有高度的序列專一性,如果其中之一的序列自然發生變化,這樣的結合就不會發生。)同樣地,這些雙股RNA又可被Dicer切割,進而產生出更多siRNA。

之後,新的siRNA或可鬆開成為單股RNA,作為引子導引RNA聚合反應的進行;或與RISC複合體聯手切割目標mRNA。學者們推測,正因為RNA干擾現象存有此擴增機制,以及另一種能於細胞間傳遞的不明機制,蟲類才得以將RNA干擾的特徵傳遞給子代。

沒有留言:

張貼留言

Related Posts with Thumbnails