Studies of Trans RNA Interference of SCA8 CTG Trinucleotide Repeats Expansion

Abstract

第八型脊髓小腦運動失調症(SCA8)和SCA8 基因3'端CTG三核苷重複擴增相關。SCA8 基因的5'端和緊鄰的KLHL1基因(actin 結合蛋白)的5'端互補。為檢視SCA8突變的異位RNA干擾效應，我們選殖、定序了包含0、23、88、157 個CTA/CTG重複的人類SCA8 cDNA及全長的KLHL1 cDNA、5'端254-bp的TBP cDNA（包含36個CAG重複），表現在人類胚胎293腎細胞，進行螢光活化細胞分檢(FACS)、RNA 螢光原位雜合(RNA FISH)分析。當GFP標記的KLHL1 cDNA與SCA8 cDNA共同表現在HEK-293細胞時，螢光活化細胞分檢分析顯示SCA8可負調節KLHL1的表現。包含CAG 重複的5'端TBP cDNA與SCA8 cDNA共同表現在HEK-293細胞時，SCA8抑制其的表現情形和SCA8重複長度相關。SCA8 RNA對KLHL1 和CAG重複RNA基因表現的抑制，在不包含CTA/CTG重複與包含157個CTA/CTG重複間有顯著差異。RNA螢光原位雜合(FISH)實驗顯示，88及157個重複CUG重複擴增的SCA8 RNA會形成核糖核酸聚焦。本實驗結果顯示SCA8的致病機轉可能和核糖核酸聚焦形成及包含KLHL1、CAG重複RNA基因表現的不正常的調控相關。

The mutation causing spinocerebellar ataxia type 8 (SCA8) has been identified as a CTG repeats expansion in the 3' region of the SCA8 gene, which overlaps the 5' region of the Kelch-like 1 (KLHL1) gene. To examine the trans RNA interference, the human SCA8 cDNA with 0, 23, 88 and 157 CTA/CTG combined repeats as well as full-length KLHL1 and 5' 254-bp CAG repeats-containing TBP cDNA were cloned and expressed in human embryonic kidney 293 cells for fluorescence activated cell sorting (FACS) and RNA fluorescent in situ hybridization (RNA FISH) analyses. FACS analysis of cells co-expressing SCA8 and GFP-tagged KLHL1 revealed that SCA8 may function as a negative regulator of KLHL1. Co-expression of SCA8 and 5' CAG repeats-containing TBP cDNA fragment also demonstrated repeat length-dependent down-regulation on the expression of CAG repeats-containing RNA. When compared to that of SCA8 transcripts without combined repeats, the suppressive activity for both KLHL1 and CAG repeats-containing cDNA fragment was significantly different for SCA8 transcripts carrying 157 combined repeats. RNA FISH experiments further revealed ribonuclear foci formation in cells carrying expanded 88 and 157 combined repeats. The results indicate that the pathogenesis of SCA8 may mediate through the ribonuclear foci formation and mis-regulation of KLHL1 and CAG repeats-containing RNA expression.