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...

Cell adhesion mediated by leukocyte integrin CR3 (CD11b/CD18, alpha m beta 2) may be rapidly modulated without changes in receptor number, and transient changes in adhesivity are thought to be driven by reversible alteration of the affinity of CR3 for ligand. Here we measure the binding affinity of CR3 using purified active and inactive receptor and the ligand, C3bi, coupled to alkaline phosphatase. Immobilized, active CR3 bound saturably and with high affinity (12.5 +/- 4.7 nM). In contrast, inactive CR3 exhibited no measurable binding. High affinity binding could be restored by the addition of the activating anti-CR3 monoclonal antibody KIM-127 to inactive CR3. Since the affinity of KIM-127 for active and inactive receptor was identical, it cannot contribute the energy to convert a low affinity receptor into a high affinity receptor. Rather, KIM-127 appears to facilitate binding of C3bi by lowering the activation energy for the shift from an inactive to an active state. These results suggest that CR3-mediated binding and detachment of cells is not driven by a reversible change in affinity but by two mechanistically distinct processes, an energetically neutral activation step for binding and an energy-dependent step that reverses binding of ligand.