Contribution of trans-splicing, 5' -leader length, cap-poly(A) synergism, and initiation factors to nematode translation in an Ascaris suum embryo cell-free system.
Trans-splicing introduces a common 5' 22-nucleotide sequence with an N-2,2,7-trimethylguanosine cap (m (2,2,7)(3)GpppG or TMG-cap) to more than 70% of transcripts in the nematodes Caenorhabditis elegans and Ascaris suum. Using an Ascaris embryo cell-free translation system, we found that the TMG-cap and spliced leader sequence synergistically collaborate to promote efficient translation, whereas addition of either a TMG-cap or spliced leader sequence alone decreased reporter activity. We cloned an A. suum embryo eIF4E homolog and demonstrate that this recombinant protein can bind m(7)G- and TMG-capped mRNAs in cross-linking assays and that binding is enhanced by eIF4G. Both the cap structure and the spliced leader (SL) sequence affect levels of A. suum eIF4E cross-linking to mRNA. Furthermore, the differential binding of eIF4E to a TMG-cap and to trans-spliced and non-trans-spliced RNAs is commensurate with the translational activity of reporter RNAs observed in the cell-free extract. Together, these binding data and translation assays with competitor cap analogs suggest that A. suum eIF4E-3 activity may be sufficient to mediate translation of both trans-spliced and non-trans-spliced mRNAs. Bioinformatic analyses demonstrate the SL sequence tends to trans-splice close to the start codon in a diversity of nematodes. This evolutionary conservation is functionally reflected in the optimal SL to AUG distance for reporter mRNA translation in the cell-free system. Therefore, trans-splicing of the SL1 leader sequence may serve at least two functions in nematodes, generation of an optimal 5'-untranslated region length and a specific sequence context (SL1) for optimal translation of trimethylguanosine capped transcripts.
Department of Biology, City University of New York Graduate Center, College of Staten Island, Staten Island, New York 10314, USA.
Sabbi
S
Lall
Cassandra C
CC
Friedman
Marzena
M
Jankowska-Anyszka
Janusz
J
Stepinski
Edward
E
Darzynkiewicz
Richard E
RE
Davis
D020121:5' Untranslated Regions / Q000737:chemistry; D000595:Amino Acid Sequence; D000818:Animals; D017165:Ascaris suum / Q000196:embryology / Q000235:genetics*; D002474:Cell-Free System; D018387:Codon, Initiator; D039561:Eukaryotic Initiation Factor-4E / Q000502:physiology*; D008969:Molecular Sequence Data; D011061:Poly A / Q000235:genetics*; D014176:Protein Biosynthesis; D012315:RNA Caps / Q000235:genetics*; D020038:RNA, Spliced Leader / Q000737:chemistry / Q000502:physiology*; D020040:Trans-Splicing
D020121:5' Untranslated Regions; D018387:Codon, Initiator; D039561:Eukaryotic Initiation Factor-4E; D012315:RNA Caps; D020038:RNA, Spliced Leader; D011061:Poly A