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Translation initiation, the first and most highly regulated step of translation, begins with several eukaryotic initiation factors (eIFs) and tRNA assembling with the small ribosomal subunit to form a pre-initiation complex (PIC). PIC formation is followed by mRNA recruitment, during which the PIC attaches to the 5' end of the mRNA, and then scans in the 3' direction to locate the AUG start codon. eIF3 is the largest, most complex eIF and is required for mRNA recruitment. To better understand the role of eIF3 in translational regulation, we are currently investigating a degron mutant of the eIF3a and eIF3b subunits (eIF3a/b Degron), which mimics the complete loss of eIF3. Previous ribosome profiling experiments identified specific mRNA transcripts whose translation efficiencies were most significantly changed with the loss of eIF3 function. However, these experiments are unable to identify the specific mRNA isoforms to which ribosome footprint reads map. We are now testing an alternative approach to investigate the role of eIF3 during translation initiation employing long-read nanopore sequencing and parallel RT-qPCR analysis of fractionated RNA from yeast extracts. We have employed polysome profiling to glean information about the translational status of mRNA within wild-type and eIF3a/b Degron cells and to separate mRNA attached to multiple ribosomes (polysomes) from mRNAs with fewer ribosomes attached. Subsequent analysis of purified total and polysome RNA by long read sequencing enables us to detect structural variants of RNA that we would be unable to capture using other sequencing methods. Additionally, we are conducting qPCR to measure relative differences in translation of specific transcripts between polysome RNA and total RNA as well as translational differences between eIF3a/b Degron and wild-type cells. This work is supported by NIH/NIGMS R15 GM140372-01.
Freedman et al. (Fri,) studied this question.