Sequences necessary for trans-splicing in transiently transfected Brugia malayi.
Many genes in parasitic nematodes are both cis- and trans-spliced. Previous studies have demonstrated that a 7nt element encoded in the first intron of the Brugia malayi 70kDa heat shock protein (BmHSP70) gene was necessary to permit trans-splicing of transgenic mRNAs in embryos transfected with constructs encoding portions of the BmHSP70 gene. Here we demonstrate that this element (the B. malayi HSP70 trans-splicing motif, or BmHSP70 TSM) is necessary and sufficient to direct trans-splicing of transgenic mRNAs derived from two genes naturally containing this motif. Mutations introduced into any position of the BmHSP70 TSM abrogated its ability to direct trans-splicing. Transgenic mRNAs derived from embryos transfected with constructs containing promoters and associated downstream domains from two normally trans-spliced genes that lack a BmHSP70 TSM homologue (the B. malayi 12kDa small subunit ribosomal protein (BmRPS12) gene and the B. malayi RNA-binding protein (BmRBP1) gene), were not trans-spliced. Transfer of the BmHSP70 TSM into the first intron of the BmRPS12 gene rendered it competent for trans-splicing. Insertion of the BmHSP70 TSM into the single intron of the BmRBP1 gene did not render it trans-splicing competent. However, tagged constructs of the full-length BmRBP1 gene were trans-splicing competent. An analysis of the first exons and introns of over 200 trans-spliced B. malayi genes found homologues for the BmHSP70 TSM in roughly 25%. Thus, while the BmHSP70 TSM is necessary and sufficient to direct trans-splicing in some genomic contexts, independent trans-splicing signals are employed by other genes.
Liu
Canhui
C
Gorgas Center for Geographic Medicine, Division of Infectious Diseases, University of Alabama at Birmingham, Birmingham, AL 35294, USA.
de Oliveira
Ana
A
Higazi
Tarig B
TB
Ghedin
Elodie
E
DePasse
Jay
J
Unnasch
Thomas R
TR
D000818:Animals; D017178:Brugia malayi / Q000196:embryology / Q000235:genetics*; D019295:Computational Biology; D005260:Female; D005786:Gene Expression Regulation; D018840:HSP70 Heat-Shock Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D009154:Mutation; D018510:RNA, Helminth / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics; D020038:RNA, Spliced Leader; D020040:Trans-Splicing; D014162:Transfection
D018840:HSP70 Heat-Shock Proteins; D018510:RNA, Helminth; D012333:RNA, Messenger; D020038:RNA, Spliced Leader