Roles and interactions of the specialized initiation factors EIF4E2, EIF4E5, and EIF4E6 in Trypanosoma brucei: EIF4E2 maintains the abundances of S-phase mRNAs.

Trypanosoma brucei has six versions of the cap-binding translation initiation factor EIF4E. We investigated the functions of EIF4E2, EIF4E3, EIF4E5, and EIF4E6 in bloodstream forms. We confirmed the protein associations previously found in procyclic forms and detected specific copurification of some RNA-binding proteins. Bloodstream forms lacking EIF4E5 grew normally and differentiated to replication-incompetent procyclic forms. Depletion of EIF4E6 inhibited bloodstream-form trypanosome growth and translation. EIF4E2 copurified only the putative RNA-binding protein SLBP2. Bloodstream forms lacking EIF4E2 multiplied slowly, had a low maximal cell density, and expressed the stumpy-form marker PAD1 but showed no evidence for enhanced stumpy-form signaling. EIF4E2 knock-out cells differentiated readily to replication-competent procyclic forms. EIF4E2 was strongly associated with a subset of mRNAs that are maximally abundant in the S-phase, and these all had decreased abundances in EIF4E2 knock-out cells. Three EIF4E2 target mRNAs are also bound and stabilized by the Pumilio domain protein PUF9. Yeast 2-hybrid results suggested that PUF9 interacts directly with SLBP2, but PUF9 was not detected in EIF4E2 pull-downs. We speculate that the EIF4E2-SLBP2 complex might interact with its target mRNAs, perhaps via PUF9, only early during G1/S, stabilizing the mRNAs in preparation for translation later in S-phase or in early G2.

An Easy-to-Use Plasmid Toolset for Efficient Generation and Benchmarking of Synthetic Small RNAs in Bacteria.

Synthetic biology approaches life from the perspective of an engineer. Standardized and de novo design of genetic parts to subsequently build reproducible and controllable modules, for example, for circuit design, is a key element. To achieve this, natural systems and elements often serve as a blueprint for researchers. Regulation of protein abundance is controlled at DNA, mRNA, and protein levels. Many tools for the activation or repression of transcription or the destabilization of proteins are available, but easy-to-handle minimal regulatory elements on the mRNA level are preferable when translation needs to be modulated. Regulatory RNAs contribute considerably to regulatory networks in all domains of life. In particular, bacteria use small regulatory RNAs (sRNAs) to regulate mRNA translation. Slowly, sRNAs are attracting the interest of using them for broad applications in synthetic biology. Here, we promote a "plug and play" plasmid toolset to quickly and efficiently create synthetic sRNAs to study sRNA biology or their application in bacteria. We propose a simple benchmarking assay by targeting the acrA gene of Escherichia coli and rendering cells sensitive toward the ß-lactam antibiotic oxacillin. We further highlight that it may be necessary to test multiple seed regions and sRNA scaffolds to achieve the desired regulatory effect. The described plasmid toolset allows quick construction and testing of various synthetic sRNAs based on the user's needs.