What is the mechanism by which mammalian ribosomes are released from the mRNA following termination of translation?

Lead Research Organisation: University of Cambridge
Department Name: Biochemistry


The strong physical, behavioural, and mental similarity of identical twins shows that it is our genetic material that largely specifies what we are. This genetic material is our DNA, which can be regarded as an exceedingly long 'tape' (e.g. an enormous video tape) with some 3 billion bits of information, coding for the numerous different types of protein which carry out most of our bodily functions. The DNA can be thought of as the 'master tape', an archive of thousands of normal length videos joined together. The process of decoding this information in the DNA involves first making a working copy of one of these videotapes in the archive. This is then decoded by a small particle known as a ribosome, which essentially carries out an analogous function to that of a video recorder (VCR) as it decodes the information as pictures. These 'biological videos' are similar to real videos in so far as the part of the tape with the pictorial information is preceded by a short leader length that has no information, and ends with a trailer that is likewise meaningless. We know that our biological VCR (the ribosome) finds the point where the true information starts by searching or scanning in fast-forward mode through the leader. However, when the ribosome-like VCR has reached the end of the pictorial information, it does not scan or try to read the meaningless trailer. Instead, as soon as the pictorial information has come to an end, the biological video is essentially ejected from the biological VCR. While we understand how this ejection mechanism works in the case of the bacterial 'VCR', we know absolutely nothing about how the mammalian eject mechanism works, except that it seems to be completely different from the bacterial mechanism. The aim of this project is to find out how the mammalian 'biological VCR' ejects the biological video cassette.

Technical Summary

It is universally agreed that when mammalian polyribosomes are incubated in the presence of an inhibitor of initiation, the ribosomes run off the mRNA (i.e. they are released from the mRNA following termination) and accumulate as 80S monomeric ribosomes. However, we know absolutely nothing about the mechanism of this release, or what protein factors are required. This represents the most significant complete gap in our understanding of the mechanism of mRNA translation in eukaryotes. It is known that in eubacteria the ribosome release is catalysed by RRF (ribosome release factor) together with elongation factor eEF2 and GTP. However, no RRF orthologue has been found in eukaryotic cytoplasm. One of the reasons why we are so ignorant of the mechanism of ribosome release from the mRNA following termination at a stop codon is that the assays for the release of the peptide by the termination factors use short oligonucleotides as mRNA-mimics, which readily dissociate spontaneously from the ribosome. One of the 3 sub-projects in this proposal will use longer RNAs (>35 nt.) with a zero-length open reading frame (e.g. ..AUGUAA..), which should remain associated with the ribosome following hydrolysis of the P-site (formyl)Met-tRNAi. Another sub-project will follow the approach that was successfully used to identify and purify eubacterial ribosome release factor (RRF). This examines the release of ribosomes from the mRNA following premature pseudo-termination by puromycin, which leaves the ribosome in precisely the same state as bona fide termination at a stop codon: an empty A-site and a deacylated tRNA in the P-site (or the P/E site in the hybrid states model). The project will examine whether ribosome release from the mRNA in either of these situations is promoted by any combination of the termination factors eRF1 and eRF3 plus eEF2 (and GTP). In the event that no combination of these factors effects ribosome release, we will examine whether post-ribosomal supernatant contains an additional protein capable of promoting such release, and, if so, we will attempt to purify any such activity and characterise it. There are indications in the literature that after puromycin-mediated pseudo-termination of translation, ribosomes may reinitiate translation close to the position on the mRNA where the puromycin intervention occurred. The implication is that either there is no ribosome release factor that can function in this situation, or that any such factor is inefficient. As a corollary of our search for a mammalian RRF, we will examine whether post-puromycin reinitiation really does occur, and, if so, we will attempt to measure the efficiency of any such in-frame reinitiation, and address the question of whether reinitiation also occurs in the other two reading frames.
Description This project addressed two important but unsolved (in 2006 when the grant commenced) issues in mammalian mRNA translation: (i) what is the mechanism by which ribosomes are released from the mRNA following termination of translation of the open-reading frame (ORF); (ii) why are mammalian ribosomes normally incapable of translating the downstream ORF of a bicistronic mRNA except when the upstream ORF is very short? The project investigated the underlying mechanism of one of the few exceptions to the latter rule: the reinitiation that occurs on the subgenomic bicistronic mRNA of feline calicivirus, a member of the norovirus family which includes (human) winter vomiting virus. Despite the fact that the upstream ORF (coding for the major virus coat protein) is very long, some 10-20% of the ribosomes translating it proceed to reinitiate translation of the downstream ORF, which codes for a minor coat protein that is essential for virus viability. This reinitiation was shown to require the terminal ~80 nucleotides of the upstream ORF, but no other viral sequences. Translation must proceed through this element and the reinitiation site AUG codon must be located in close proximity downstream of it. The 80-nucleotide element was shown to bind 40S ribosomal subunits and initiation factor eIF3; mutants defective in reinitiation showed reduced affinity for eIF3 and/or defective 40S subunit binding. These findings dovetail nicely with those of Pisarev AV, Hellen CU and Pestova TV (2007. Cell 131:286-299), who showed that initiation factors eIF1, eIF1A and eIF3 can promote post-termination disassembly and release of ribosomes from mRNA. Our results suggest a mechanism of reinitiation in which some of the eIF3/40S complexes formed during disassembly of post-termination ribosomes at the end of the upstream ORF bind to the 80-nucleotide element in a position appropriate for reinitiation following acquisition of an eIF2/GTP/Met-tRNAi ternary complex.
In a further development of this work, a collaboration with Dr. Ian Brierley, Dept of Pathology, University of Cambridge (who was supported by BB/C007034/1) showed that a virtually identical reinitiation mechanism operates on the bicistronic mRNA encoded by segment 7 of (human) influenza B viruses. In particular, reinitiation in this case has an absolute requirement for a short element at the end of the upstream ORF, and this critical element has an essential UGGA motif in both the caliciviruses and influenza B viruses.
Exploitation Route Our results obtained with the feline calicivirus subgenomic mRNA system have been taken forward by Dr. Ian Brierley (Dept of Pathology, University of Cambridge), who found a very similar (but not quite identical) mechanism of reinitiation in the bicistronic mRNA encoded by segment 7 of the influenza B viruses. It would be quite straightforward to apply the methods and approaches that we (and others) have developed to study reinitiation in the calicivirus and influenza virus system, to examine other putative cases of reinitiation on bicistronic cellular or viral mRNAs.
Sectors Healthcare,Pharmaceuticals and Medical Biotechnology

Description Although the findings of work supported by this grant have been influential on subsequent academic research (as described under Key Findings), they have not yet had any known influence outside academia.
Sector Healthcare,Pharmaceuticals and Medical Biotechnology