Establishment of an in vitro trans-splicing system in Trypanosoma brucei that requires endogenous spliced leader RNA.
In trypanosomes a 39 nucleotide exon, the spliced leader (SL) is donated to all mRNAs from a small RNA, the SL RNA, by trans-splicing. Since the discovery of trans-splicing in trypanosomes two decades ago, numerous attempts failed to reconstitute the reaction in vitro. In this study, a crude whole-cell extract utilizing the endogenous SL RNA and synthetic tubulin pre-mRNA were used to reconstitute the trans-splicing reaction. An RNase protection assay was used to detect the trans-spliced product. The reaction was optimized and shown to depend on ATP and intact U2 and U6 snRNPs. Mutations introduced at the polypyrimidine tract and the AG splice site reduced the reaction efficiency. To simplify the assay, RT-PCR and quantitative real-time PCR assays were established. The system was used to examine the structural requirements for SL RNA as a substrate in the reaction. Interestingly, synthetic SL RNA assembled poorly to its cognate particle and was not utilized in the reaction. However, SL RNA synthesized in cells lacking Sm proteins, which is defective in cap-4 modification, was active in the reaction. This study is the first step towards further elucidating the mechanism of trans-splicing, an essential reaction which determines the trypanosome transcriptome.
Shaked
Hadassa
H
The Mina & Everard Goodman Faculty of Life Sciences, and Advanced Materials and Nanotechnology Institute Bar-Ilan University, Ramat-Gan 52900, Israel.
Wachtel
Chaim
C
Tulinski
Pawel
P
Yahia
Nasreen Hag
NH
Barda
Omer
O
Darzynkiewicz
Edward
E
Nilsen
Timothy W
TW
Michaeli
Shulamit
S
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D006358:Hot Temperature; D009154:Mutation; D016133:Polymerase Chain Reaction; D012315:RNA Caps / Q000378:metabolism; D012322:RNA Precursors / Q000737:chemistry; D022821:RNA Splice Sites; D012333:RNA, Messenger / Q000737:chemistry; D012342:RNA, Small Nuclear / Q000378:metabolism; D020038:RNA, Spliced Leader / Q000096:biosynthesis / Q000378:metabolism*; D020040:Trans-Splicing; D014346:Trypanosoma brucei brucei / Q000235:genetics* / Q000378:metabolism
D012315:RNA Caps; D012322:RNA Precursors; D022821:RNA Splice Sites; D012333:RNA, Messenger; D012342:RNA, Small Nuclear; D020038:RNA, Spliced Leader; C405484:U2 small nuclear RNA; C405486:U6 small nuclear RNA; D000255:Adenosine Triphosphate