Writing an introduction: Added Value Talk for
Virology Students 5/13/2013
Anne Simon
Introduction
Regulation of gene expression is an essential cellular process that occurs
at multiple levels including translation of mRNAs into proteins.
Most regulation at translation level is exerted at the translation initiation
stage where the AUG start codon is identified and decoded by the
methionyl tRNA specialized for initiation (1, 2).
Canonical translation initiation in eukaryotes is a complex, multistep
process in which 5’m7GpppN cap and 3’ poly(A) tail on a transcribed
mRNA cooperate together to recruit translation initiation factors and
40S ribosomal subunits to form the preinitiation complex (PIC).
Then the preinitiation complex (PIC) scans the message in a 5’ to 3’
direction and recognizes the AUG initiation codon where 60S ribosomal
subunits join to form the 80S ribosome–mRNA complex to start the
translation.
Cellular gene expression is regulated at multiple steps including when
mRNAs are translated into proteins.
Post-transcriptional control of gene expression can occur during
translation initiation, when the correct start codon is identified and
decoded [1,2].
In eukaryotes, canonical translation initiation is a complex, multistep
process in which the 5’m7GpppN cap and 3’ poly(A) tail cooperate to
recruit translation initiation factors and ribosomal subunits.
The 5’ cap is recognized by the cap-binding protein eIF4E, which
along with scaffold protein eIF4G is a subunit of the eukaryotic
translation initiation factor complex eIF4F [3].
Simultaneous binding of eIF4G to eIF4E and poly(A)-binding protein
forms a bridge that circularizes the mRNA, which is thought to recycle
ribosomes leading to more efficient translation [4].
The 43S ribosome preinitiation complex (PIC), consisting of the 40S
ribosomal subunit and met-tRNA-containing ternary complex, is
recruited to the 5’ end of mRNA via the interaction between eIF4G and
additional initiation factors [3,5].
The PIC scans the message in a 5’ to 3’ direction until contacting an
initiation codon in the proper context. The 60S ribosomal subunit then
joins to form the 80S ribosome–mRNA complex, and translation
elongation commences with the entry of the appropriate aminoacylated
tRNA complex into the ribosome A-site.
Nonconventional mechanisms to translation are operative during cellular
stress or utilized by atypical mRNAs that lack the cap structure at the
5’end or the poly(A) tail at the 3’end (1, 3).
This mechanism depends on specific highly structured cis-acting RNA
sequences called internal ribosome entry sites (IRESes) which are found
in a broad range of RNA viruses and in some eukaryotic cellular
mRNAs (4).
IRESs are known to attract eukaryotic ribosomal translation initiation
complex and thus promote translation initiation independently of the
presence of the commonly utilized 5’m7GpppN cap structure.
In addition to IRES, eukaryotic mRNAs can contain upstream open
reading frames (uORFs), which allows a high frequency of reinitiation
by post-termination 40S subunits or translation regulatory elements
located in 3’UTR.
Nonconventional mechanisms of translation operate during cellular
stress and are also utilized by mRNAs lacking a 5’ cap and/or a 3’
poly(A) tail [1,6].
Cap-independent ribosome entry is frequently studied using animal
RNA viruses that have replaced the 5’ cap with highly structured, cisacting elements known as internal ribosome entry sites (IRESs) that
encompass or are near the initiation codon [7].
Attraction of the small ribosomal subunit to different viral IRES usually
requires a subset of initiation factors but can also occur in the absence
of factors [8].
Cap-independent translation elements (CITEs) in the 3’UTR is much
more widespread in plant RNA viruses.
Although the sequence and structure of plant virus 3’cap-independent
translation enhancers (3’CITEs) diverse significantly, most of them
involve in bridging 5’ UTR and 3’UTR by long-distance kissing-loop
interactions, which is thought to deliver bound translation initiation
factors to the 5’-terminal of viral RNA to mediate initiation of
translation.
In contrast with animal RNA viruses, plant RNA viruses whose
genomes lack a 5’ cap and 3’ poly(A) tail have short 5’UTR that do not
contain similar highly structured IRES elements.
Translation of plant RNA viruses in the family Tombusviridae requires
distinctive cap-independent translation elements (CITEs) located within
the viral genomic RNA’s 3’ UTR and nearby coding region [9].
3’CITEs are currently divided into at least eight classes based mainly
on their secondary structures, including Y-shaped, I-shaped, BTE-like,
and TED-like [10].
3’CITEs are bound by various translation initiation factors and are
generally associated with a long-distance, kissing-loop interaction that
connects the 3’CITE with the 5’ UTR, resulting in genome
circularization that delivers the bound factors to the viral 5’end [11-14].
How and when the 3’ CITE-bound factors contribute to ribosome
recruitment, and how ribosomes are delivered at or near the 5’ terminus
before scanning to the initiation codon [15-17], is not known.
Transfer RNAs (tRNA) are the adaptor molecules that serve as the
physical link between the nucleotide sequence of nucleic acids and the
amino acid sequence of proteins by carrying specific amino acids to the
protein synthetic machinery as directed by a three-nucleotide codons in
a messenger RNA (mRNA).
In addition to a critical role translating mRNAs into proteins, tRNAlike structures also exist internally or at the 3' termini of genomes of a
growing number of RNA viruses that have a variety of functions
including being used as 3’CITEs.
In addition to their major role in protein biosynthesis, tRNAs
participitates in a variety of cellular roles, such as amino acid
biosynthesis (5), transcriptional regulation (6), viral packaging (7) and
viral genome replication (8, 9).
Some capped, positive-strand RNA plant viruses including tymo-,
bromo, and tobamoviruses, contain 3’ terminal tRNA-like structures
(TLS) that are aminoacylated by host aminoacyl tRNA-synthetases and
function in replication, translation, packaging, and fidelity of the
genome (7-14).
Many viruses has evolved to utilize elements that mimic of tRNA in
terms of the structure or biochemical properties to perform various
fundamental roles in viral life cycles.
Transfer RNA-like structures (TLSs) are functional mimics of tRNAs
that are found at the 3’ end of the genomes of a number of plant positive
strand RNA plant viruses.
TLSs have been found capable of valylation, tyrosylation or
histidylation at the viral RNA’s 3’terminal CCA by a host cell
aminoacyl tRNA-synthetase (AARS), which are represented by turnip
yellow mosaic virus (TYMV) TLS, brome mosaic virus (BMV) TLS
and tobacco mosaic virus (TMV) TLS respectively (10-12).
The roles of TLSs vary widely among different viruses including
regulation of translation, replication of the genome, fidelity of the 3’
end of the viral RNA and encapsidation of viral RNAs (13, 14).
A tRNA anti-codon-like element (TLE) in the HIV-1 genome has been
recently reported to mimics the anti-codon loop of tRNALys to increase
the efficiency of tRNALys3 annealing to viral RNA which is crucial for
viral replication.
Other tRNA mimicry in RNA viruses includes the intergenic region
(IGR) IRES region of cricket paralysis virus, which interacts with the
ribosome’s decoding groove by mimicing the tRNA anticodon-mRNA
codon interaction (15).
The retrovirus HIV-1 contains an internal tRNA anticodon stem-like
element that mimics the anti-codon loop of tRNALys and functions to
increase the efficiency with which tRNALys3 anneals to the viral
genome during replication [20].
Other examples of tRNA mimicry in RNA viruses include a TLS
replication enhancer in the RNA3 intergenic region of Brome mosaic
virus and related cucomoviruses, which is a substrate for tRNA
modification enzymes in vivo [21], and the IRES of Cricket paralysis
virus, which interacts with the ribosome’s decoding groove by
mimicking the tRNA anticodon-mRNA codon interaction [22].
Rare examples of tRNA mimicry have also been reported in 3’ CITE of
plant RNA viruses.
An unique 3’CITE discovered in the 3’ UTR of carmovirus Turnip
crinkle virus (TCV) folds into a T-shaped structure (TSS) and it is
capable of binding to the P-site of 80S ribosomes and 60S ribosomal
subunits, which is indispensable for the CITE activity (16-18).
It has been suggested recently that circularization of the viral genome of
TCV may be through the simultaneous binding of the 40S ribosomal
subunit to a pyrimidine-rich sequence in the 5’UTR and the 60S subunit
to the 3’UTR TSS (19).
Our previous work has identified an 81-nt tRNA-shaped element (klTSS) near the center of the 3’UTR of Pea enation mosaic virus (PEMV)
RNA2 that enhances translation by binding to ribosomes/40S/60S
subunits, and engaging in a long distance RNA:RNA kissing-loop
interaction with a 5’ coding region hairpin.
A translation element just downstream from the kl-TSS (the PTE) binds
to eIF4E and is needed for full activity of the kl-TSS (20, 21).
An internally located tRNA-mimic that forms from three hairpins and
two pseudoknots functions as a 3’CITE in the carmovirus Turnip
crinkle virus (TCV) (family Tombusviridae)
[23,24].
The TCV TSS binds to 80S ribosomes and 60S ribosomal subunits in
the vicinity of the P-site [23] and is not associated with any longdistance kissing-loop interaction.
Rather, circularization of the genome may result from 60S subunits
bound to the 3’UTR TSS joining 40S subunits bound to a pyrimidinerich sequence in the 5’UTR [25].
A second tRNA mimic that functions as a 3’CITE was recently
discovered in the 3’UTR of Pea enation mosaic virus (PEMV).
PEMV is a bipartite virus with two single-stranded plus-sense RNAs
that were originally independent viruses.
PEMV RNA 2 (will be referred to throughout as PEMV) is classified
as an umbravirus and is missing the 5’ cap and poly(A) tail, similar to
viruses in the Tombusviridae.
PEMV is a recombinant virus of 4.2 kb, encoding a carmovirus-like
RNA-dependent RNA polymerase (RdRp) and two overlapping,
movement-associated proteins (p26, p27) from a second, unknown
virus [26] (Fig. 1).
PEMV replicates independently in protoplasts, but requires products
produced by the associated viral RNA for encapsidation and
transmission from plant-to-plant [27].
The central region of the PEMV 3’UTR (702 nt) contains a 3’CITE
known as a Panicum mosaic virus (PMV)-like enhancer, or PTE [28]
(Fig. 1).
The PEMV PTE binds to eIF4E and binding efficiency correlates with
translational enhancement [29,30].
Although PTE found in the 3’UTR of seven carmoviruses are known or
postulated to engage in long-distance kissing-loop interactions with
hairpins in the 5’UTR or nearby coding sequences [31], the PEMV PTE
relies on an adjacent, upstream element for relocalization of itself and
bound initiation factors to the 5’ end [11].
The upstream element contains a two hairpin, three-way branched
secondary structure with the 5’ side hairpin (3H1) participating in a
kissing-loop interaction with a 12-bp hairpin (5H2) located at positions
60-88 near the 5’ end of the p33 ORF (Fig. 1).
This 3’UTR branched element is predicted to fold into a TSS and is
therefore designated as a “kissing-loop TSS” (kl-TSS) [11].
Similar to the TCV TSS, the PEMV kl-TSS binds to 80S ribosomes and
60S ribosomal subunits; however, unlike the TCV TSS, the kl-TSS also
binds to 40S subunits [11].
Since both ribosome binding and long-distance RNA:RNA interaction
activities of the kl-TSS are important for efficient translation, the klTSS has been designated as a 3’CITE.
In this work we provide evidence that binding of the kl-TSS to
ribosomes and to the 5’89-nt of PEMV is mutually compatible,
suggesting a model whereby the RNA:RNA interaction helps to
recycle ribosomes/ribosomal subunits that bind to the kl-TSS
located in cis to enhance re-initiation of translation at the 5’ end.
In this report, we find that the kl-TSS and TCV TSS occupy different
sites in the 80S ribosome and that the kl-TSS can simultaneously bind
to ribosomes and interact with the 5’ hairpin, leading to a re-evaluation
of the orientation of the kl-TSS with respect to canonical tRNAs.
We also report that the kl-TSS inhibits translation more efficiently than
the associated PTE when added to a reporter template in trans, and that
both RNA:RNA interaction and ribosome binding activities contribute
to translational inhibition.
These results suggest: (i) the PTE may be contributing to kl-TSS
ribosome binding and is not associated with an independent function;
(ii) RNA viruses have evolved at least two different mechanisms for
using TSS-type 3’CITEs; and (iii) the RNA:RNA interaction of the klTSS can directly place bound ribosomes/ribosomal subunits at the 5’
end for re-initiation of translation.