The role of 5 '-leader length, secondary structure and PABP concentration on cap and poly(A) tail function during translation in Xenopus oocytes

The 5'-cap structure and poly(A) tail of eukaryotic mRNAs function synergis tically to promote translation initiation through a physical interaction be tween the proteins that bind to these regulatory elements, In this study, w e have examined the effect of leader length and the presence of secondary s tructure on the translational competence and the function of the cap and po ly(A) tail for mRNAs microinjected into Xenopus oocytes, Increasing the len gth of the 5'-leader from 17 to 144 nt resulted in a 2- to 4-fold increase in expression from an mRNA containing an unstructured leader but increased expression up to 20-fold for an mRNA containing 5'-proximal structure. Cons equently, the presence of secondary structure was less inhibitory for those mRNAs with a longer 5'-leader. Go-injection of poly(A)-binding protein (PA BP) mRNA increased the function of the cap and poly(A) tail in promoting tr anslation from poly(A)(+) but not poly(A)(-) mRNAs, particularly for mRNAs containing secondary structure. In the absence of an internal ribosome entr y site, expression from the distal cistron of a dicistronic mRNA increased as a function of the length of the intercistronic region and the concentrat ion of PABP, The inhibitory effect of intercistronic located secondary stru cture on translation was position-dependent. Indeed, the effect of secondar y structure was abolished if positioned 134 nt upstream of the distal cistr on, These data suggest that the length of a leader, the presence of seconda ry structure and the concentration of PABP determine the extent to which th e cap and poly(A) tail regulate translation.