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.
Lall
Sabbi
S
Department of Biology, City University of New York Graduate Center, College of Staten Island, Staten Island, New York 10314, USA.
Friedman
Cassandra C
CC
Jankowska-Anyszka
Marzena
M
Stepinski
Janusz
J
Darzynkiewicz
Edward
E
Davis
Richard E
RE
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