Riboproteomic Approaches to Understanding IRES Elements - Biophysical Approaches to Translational Control of Gene Expression - page 101

PCBP1-2 Poly(rC) binding protein

PTB Polypyrimidine tract-binding protein

PV Poliovirus

RRM RNA recognition motif

SILAC Stable isotopic labeling with amino acid in cell culture

Unr Upstream of N-ras

6.1 Introduction: IRES Elements and Their

Trans -Acting Factors

The process of RNA translation consists of sequential steps, initiation, elongation,
termination, and ribosome recycling. In eukaryotes, translational control is mainly
exerted at the initiation step, assisted by proteins termed translation initiation fac-
tors (eIFs). Most eukaryotic mRNAs initiate translation using a cap-dependent
mechanism, by which the m 7 GpppN (cap) at the 5¢ end of the mRNA is recognized
by the eIF4F complex (consisting of the cap-binding factor eIF4E, the RNA heli-
case eIF4A, and the scaffold protein eIF4G that in turn interacts with eIF4E, eIF4A,
eIF3, and the poly(A) binding protein (PABP1)). The pre-initiation complex scans
in 5¢ to 3¢ direction until an appropriate initiation codon is encountered, leading to
the 48S complex formation assisted by eIF1, eIF2, and eIF5. Finally, eIF5B medi-
ates joining of the 60S and 40S ribosomal subunits, generating the 80S complex
competent for protein synthesis.
In contrast, initiation of protein synthesis in mRNAs translated under stress con-
ditions is often driven by internal ribosome entry site (IRES) elements. IRES are
cis -acting sequences usually located in the 5¢ untranslated region (UTR) of mRNAs,
that recruit the translation machinery using a 5¢ end-independent mechanism with
the help of eIFs and RNA-binding proteins termed IRES transacting factors (ITAFs).
The process of internal initiation operates in RNA viruses as well as in cellular
mRNAs translated under stress conditions that compromise cap-dependent transla-
tion (Martinez-Salas et al. 2008 ; Spriggs et al. 2010 ). However, there is great diver-
sity regarding their primary sequences, secondary structures, and ITAFs required
for activity. The large degree of diversity within viral IRES is typically illustrated by
two distantly related IRES elements, the one present in the intergenic region of the
dicistroviruses genome, whose activity depends exclusively on its three-dimensional
RNA structure, and those present in the genome of picornaviruses that have
significantly longer nucleotide sequences and depend on both RNA structure and
ITAFs (Filbin and Kieft 2009 ; Martinez-Salas 2008 ). It is thought that RNA-binding
proteins assist picornavirus IRES activity through their involvement in the acquisi-
tion of a proper RNA structure.
The lack of apparent conserved features has led to the view that different IRES
elements could recruit the ribosomal subunits assisted by unique sets of ITAFs.
Thus, understanding the role played by ITAFs is crucial to unravel the strategies
employed by mRNAs to recruit the translation machinery under conditions of