Functional Characterization of Five eIF4E Isoforms inCaenorhabditis elegans

Abstract

Recognition of the 5-cap structure of mRNA by eIF4E is a critical step in the recruitment of most mRNAs to the ribosome. In Caenorhabditis elegans, ϳ70% of mRNAs contain an unusual 2,2,7-trimethylguanosine cap structure as a result of trans-splicing onto the 5 end of the pre-mRNA. The characterization of three eIF4E isoforms in C. elegans (IFE-1, IFE-2, and IFE-3) was reported previously. The present study describes two more eIF4E isoforms expressed in C. elegans, IFE-4 and IFE-5. We analyzed the requirement of each isoform for viability by RNA interference. IFE-3, the most closely related to mammalian eIF4E-1, binds only 7-methylguanosine caps and is essential for viability. In contrast, three closely related isoforms (IFE-1, IFE-2, and IFE-5) bind 2,2,7-trimethylguanosine caps and are partially redundant, but at least one functional isoform is required for viability. IFE-4, which binds only 7-methylguanosine caps, is most closely related to an unusual eIF4E isoform found in plants (nCBP) and mammals (4E-HP) and is not essential for viability in any combination of IFE knockout. ife-2, ife-3, ife-4, and ife-5 mRNAs are themselves trans-spliced to SL1 spliced leaders. ife-1 mRNA is transspliced to an SL2 leader, indicating that its gene resides in a downstream position of an operon.Eukaryotic mRNAs and small nuclear RNAs synthesized by RNA polymerase II are posttranscriptionally modified to form a 5Ј-5Ј GpppN linkage (1). The 5Ј-terminal G is methylated at N7 while still in the nucleus to yield an MMG 1 cap. The cap of small nuclear RNAs is then further methylated at N2 in the cytoplasm to yield a TMG cap (2). Methylation of small nuclear RNAs is dependent upon the binding of Sm proteins to form small nuclear ribonucleoproteins. Formation of the TMG cap is the targeting signal for import of small nuclear ribonucleoproteins back into the nucleus to take part in pre-mRNA splicing (3, 4). mRNAs, on the other hand, which possess only the MMG cap, remain in the cytoplasm.In some primative eukaryotes, including Caenorhabditis elegans, mRNAs acquire a TMG cap through the process of trans-splicing (5). Primary transcripts from approximately 70% of protein-coding genes are trans-spliced to 22-nt SL sequences, such that the original MMG caps are replaced with the TMG caps from the SL small nuclear RNAs (6, 7). Also common in C. elegans is the organization of genes into operons that are transcribed from a single promotor into a polycistronic RNA (8). trans-Splicing results in the processing of these primary transcripts into monocistronic mRNAs. Generally, the mRNA from the first cistron is trans-spliced to SL1,whereas mRNAs from downstream cistrons are trans-spliced to SL2 or SL2 variants (8). mRNAs that are not trans-spliced retain the original MMG cap. Thus, both MMG-and TMG-capped mRNAs are found in the cytoplasm of C. elegans. Both types of mRNA enter polyribosomes and are translated, indicating that they are competent to interact with the translational machinery (9).The recruitment of mRNAs to ribosomes is catalyzed by...

Structural Insights into Parasite eIF4E Binding Specificity for m7G and m2,2,7G mRNA Caps

“…Thus, schistosome eIF4E has relatively high affinity for the m 2,2,7 G cap. Unlike other trans-splicing organisms examined (22,23,50,64), only a single eIF4E isoform is identifiable in schistosomes. This suggests that GpppG/AAlthough the schistosome eIF4E has limited sequence identity with other eIF4Es, the overall structure in complex with either m 7 GpppG or m 7 GpppA is very similar to that observed for other eukaryotic eIF4Es (Fig.…”

“…2C). In previous eIF4E structures with a dinucleotide cap, the second base of the cap was only well resolved in the human eIF4E complex structure (22,23) apparently due to the flexibility of this nucleotide. In the human structure, the second base (m 7 GpppA) points to the C-terminal flexible loop and has some hydrogen bonding with the residues located in this loop.…”

“…Approximately 70% of mRNAs in the nematode Caenorhabditis elegans undergo trans-splicing. Several isoforms of C. elegans eIF4E and an eIF4E from the parasitic nematode Ascaris suum recognize both monomethyl-and trimethylguanosine caps to a similar extent (19,(22)(23)(24). The unique ability of transsplicing worms to interact efficiently with the m 2,2,7 G cap compared with vertebrate eIF4E represents a potential drug target against a diverse group of parasitic worms important in human and veterinary medicine.…”

Trans-spliced leader addition to mRNAs in a cnidarian

“…The presence of TMG caps on mRNAs as the result of an organism acquiring SL addition requires that the organism also evolve eIF4e proteins that can bind to TMG caps. C. elegans contains five genes encoding eIF4e isoforms (41). Three of these isoforms are closely related and bind TMG caps in addition to the monomethyl guanosine (MMG) caps, which are present on non-SL-containing mRNAs.…”

Two Arabidopsis Loci Encode Novel Eukaryotic Initiation Factor 4E Isoforms That Are Functionally Distinct from the Conserved Plant Eukaryotic Initiation Factor 4E

“…Caenorhabditis elegans has four isoforms involved in differentiation between mono-and trimethylated mRNA caps (Keiper et al, 2000) and have specialized roles for regulation of certain sets of mRNAs, particularly in the germline (Amiri et al, 2001;Song et al, 2010). Trypanosoma brucei has four isoforms with varying ability to bind cap analog and eIF4G isoforms (Freire et al, 2011).…”