Studying sequence effects of mRNA 5' cap juxtapositions on translation initiation rate using randomization strategy of the extreme 5' end of mRNA
University College Cork
Translation initiation is a complex process. The efficiency of translation initiation is determined not just by activity and availability of the translation initiation apparatus, but also the properties of mRNA 5’transcript leaders (5’TL). In most cases of cap-dependent translation, translation initiation begins with the formation of the preinitiation complex (PIC) loading and accommodation onto the m7G capped 5’end of mRNA, facilitated by m7G cap – eIF4F interactions. The PIC accommodation onto the 5’end of mRNA is a point of control in translation initiation where the role of 5’ cap proximal mRNA sequence determinants are poorly understood. To explore the effect of the nucleotides in the extreme 5’ end of mRNA on translation initiation, a library of mRNA molecules was synthesized containing all possible permutations of the first 10 nucleotides, referred to as E5S (Early 5' Sequence). The library was transfected into HEK293T cells. The lysates obtained from transfected cells were separated on a sucrose density gradient to isolate mRNAs bound to polysomes. Based on the assumption that efficiently translated mRNAs are associated with polysomes, the effect of E5S on translation initiation was measured by comparing frequencies of nucleotides (and their combinations) at specific positions in E5S from mRNAs in polysome fractions to their frequencies in E5S of the original library using massively parallel sequencing. The second position of E5S was found to have a markedly higher influence on translation initiation than positions further downstream (for technical reasons it was not possible to estimate the influence of the first position of E5S). In this position G was the most enriched nucleotide, and U was the most depleted nucleotide. Analysis of available ribosome profiling datasets did not reveal a significant association between E5S and ribosome footprint densities at the coding regions. While this work clearly suggests the influence of nucleotide context on translation initiation, it is possible that such as uORFs and RNA secondary structures, have a higher influence on translation initiation than E5S. The E5S is a previously unappreciated determinant of translation initiation, and this work suggests that differences in mRNA 5' end accessibility defined by the cap proximal sequence may be an important determinant in modulating the rate of translation initiation.
Pai, A. 2018. TStudying sequence effects of mRNA 5' cap juxtapositions on translation initiation rate using randomization strategy of the extreme 5' end of mRNA. PhD Thesis, University College Cork.