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Paola Londei - Translational regulation: from the archaea to the eukarya
of assays so far performed, including interaction
with the β and γ subunits to form the intact trimer
and capacity of binding met-tRNAi. To investigate
in vivo phosphorylation, we have performed
immunoprecipitation assays with anti α-subunit
antibodies on whole cell lysates, followed by western
blotting with anti-phosphoserine antibodies.
Preliminary results indicate that the a/eIF2 α-subunit
is indeed phosphorylated in vivo, and that the
amount of phosphorylation increases following
heat-shock, indicating that the modification may
inhibit protein synthesis. Work is in progress to
confirm and expand these results and to elucidate
the underlying molecular mechanism.
A still mysterious protein: IF6
The factor known as aIF6 (archaea) and eIF6
(eukarya) is a monomeric protein of about 25 kDa.
Eukaryotic IF6 was originally described as a ribosome
anti-association factor, which interacted with
the large ribosomal subunits inhibiting the formation
of active 80S ribosomes. Later experiments revealed
that depletion of eIF6 in yeast depressed the biosynthesis
of 60S ribosomal subunits, but had otherwise
no specific effect on translation initiation, assigning
to the factor a main role in ribosome synthesis.
Further experiments in mammals, however, have
confirmed a role for eIF6 in translational regulation,
that may or may not co-exist with other functions.
60S ribosomal subunit must release eIF6 to enter the
translation cycle; eIF6 dissociation from the ribosome
is triggered by the phosphorylation of the factor
carried out by certain regulatory kinases. More
12
recently, it has been shown that heterozygous knockout
mice for eIF6 have defective translational initiation.
Finally, it has been reported that eIF6 depletion
inhibits miRNA-induced gene silencing in both
human cells and in nematodes. Despite the many
functions reported, the mechanism of action of eIF6
in any of them remains unknown.
Archaeal IF6 shares a high degree of homology with
eIF6, both in primary sequence and in three-dimensional
structure. Our recent work has revealed that S.
solfataricus aIF6 has a ribosome anti-associating
activity and is a potent inhibitor of protein synthesis
in vitro. We have mapped for the first time the ribosomal
binding site of aIF6: it lies in the centre of the
30S-contacting face of the large ribosomal subunit.
Our model of the aIF6/50S complex reveals that the
factor inhibits the formation of the 70S ribosome by
masking a number of RNA and protein determinants
that bridge the two subunits. These data confirm the
identity of IF6 as a translation factor and rationalize
its anti-associating activity in molecular detail.
Work is in progress to unravel the mechanism whereby
aIF6 dissociates from the 50S subunit. Preliminary
data indicate that this may entail a novel type of protein
modification, possibly in association with a ribosome-dependent
GTPase activity, yet to be identified.
Selected publications
Benelli D, Marzi S, Mancone C, Alonzi T, La Teana
A, Londei P. Function and ribosomal localization of
aIF6, a translational regulator shared by archaea and
eukarya. Nucleic Acids Res. 2008, Epub.