SL Trans-Splicing in a Caenorhabditis elegans In Vitro Extract
- 1 Department of Biochemistry and Molecular Genetics, University of Colorado Denver, Anschutz Medical Campus, Aurora, Colorado 80045, USA
- 2 Department of Molecular, Cellular, and Developmental Biology, University of Colorado, Boulder, Colorado 80309, USA
- 3 Epicentre Biotechnologies, Madison, Wisconsin 53713, USA
- ↵4Corresponding author (tom.blumenthal{at}colorado.edu).
INTRODUCTION
Caenorhabditis elegans is one of a number of organisms in which spliced leader (SL) trans-splicing contributes to the maturation of many pre-mRNA transcripts. C. elegans and related nematodes have two distinct classes of SL RNA—SL1 and SL2—that are used differentially for different classes of pre-mRNAs; namely, SL1 trans-splices non-operon genes and first genes in operons, while SL2 splices downstream operon genes. In vitro RNA splicing has previously been used to analyze substrate RNA sequences, required protein and RNA factors, and splicing mechanisms in a variety of systems. This protocol describes an in vitro splicing assay using C. elegans extract that promotes both cis- and trans-splicing of substrate RNAs. It has the ability to correctly specify and detect SL1 versus SL2 trans-splicing. Operon-derived substrates splice SL2, while substrates with an intron-like sequence followed by a 3′ trans-splice site (outron) splice SL1. Unlabeled T7-transcribed RNA substrates are incubated with crude embryonic extract and trans-spliced by endogenous SL small nuclear ribonucleoproteins (snRNPs). Spliced RNA is analyzed using a reverse transcriptase polymerase chain reaction (RT-PCR). Specific RT and PCR primers ensure detection of trans-spliced substrate RNA and allow a means to distinguish between SL1 and SL2 trans-spliced products. Substrates containing introns are also cis-spliced. All reaction products are ATP-dependent.
