To compete with the host mRNAs for the cellular translation machinery and to circumvent host regulation, many viral mRNAs rely on alternative mechanisms for gene
expression. The uncapped, non-polyadenylated Barley yellow dwarf virus RNAs utilize a
novel mechanism for the recruitment of the ribosomes to the mRNA that differs from cap- dependent translation and known IRESs by its combination of (i) cap-independence, (ii) location of the translation element in the 3’ UTR, and (iii) requirement for scanning from the 5’ end of the mRNA.
In this dissertation research, I focused on the molecular mechanics of the long distance 5’-3’ interaction that is indispensable for BTE-mediated cap-independent translation. This interaction is proposed to bring the BTE into close proximity with the 5’ UTR for the delivery of the translation factors recruited at the 3’ end. My research has revealed that this kissing interaction is dynamic, switching between closed and open conformations, and perfectly coordinates - from its position from the 5’ end - initiation and regulation of gene expression. I observed that the 43S ribosomal complex is capable of entering at the 5’ end of the BYDV mRNA, but its efficiency is strongly promoted by the BTE, which maximizes the pool of factors at the 5’ end of the mRNA. In summary, 3’ BTE- mediated translation is dependent upon:
(i) The recruitment of the translation factors by the BTE in cooperation with
the long distance 5’-3’ communication. The core cap-independent translation activity of the BTE can be separated from its role in 5’
interaction. However disruption of either event abolishes 3’ end-mediated translation (Chapter 2).
(ii) The 5’ end-dependent scanning of the 5’ UTR to reach the initiation
codon, just like in cellular mRNAs (Chapter 2).
(iii) The distance between the 5’ terminus and the BTE complementary kissing
stem-loop (BCL). Reduced translation is observed when additional unstructured sequences are added to the extreme 5’ end of the mRNA, moving the BCL further downstream of the 5’ end (Chapter 2). On the other hand, the relocation of the BCL to the very 5’ end of the gRNA supports translation at a level similar at wild type (Chapter 2). However, the position of the BCL relative to the 5’ end modulates sensitivity to translation inhibition by sgRNA2 (Chapter 3) or under condition of low factor availability (Chapter 4). As revealed in Chapters 3 and 4, BYDV has evolved a unique mechanism to allow regulation of translation by harboring the BCL at different positions within its gRNA and sgRNAs (at at the extreme 5’ end at the sgRNA1, further downstream position in the gRNA, and absent in sgRNA2).
(iv) The stability of the 5’ UTR secondary structure, which becomes rate
limiting under low factor availability. This is consistent with the simple scanning efficiency mechanism. Given the evidence that BTE-mediated translation requires 5’ end–dependent ribosome scanning, a longer 5’ UTR tract with significant structure may require more translation factors in order facilitate ribosome scanning (Chapter 4).
Why a 3’ translation element?
The presence of the translation element in the 3’ UTR of the viral RNA confers several advantages: (i) it prevents translation initiation on 3’-truncated, incompletely synthesized or degraded RNAs, (ii) it ensures the presence of the translation element on the coding subgenomic RNAs, and (iii) may facilitate the switch from translation to replication as the cycle of infection progresses. The 3’ UTR harbors the cis-acting elements required both for translation and replication (2), and thereby prevents theses two processes from occurring simultaneously on the same RNA. This mechanism of 3’ cap-independent translation element is more common than one thought, as an increasing number of new translation elements continues to be identified.
Future prospects
3’ translation element and factor recognition: The basic steps of 3’ CITE-mediated translation have been delineated: they involve recruitment of the translation factors and communication with the 5’ UTR (3). However, many questions remain regarding this simplistic model. There is no prototype sequence or structure shared by all 3’ CITEs, and recent studies have revealed differences in their requirements for translation factors and in the process for the assembly of the scanning machinery. For instance, the STNV translation enhancer domain (TED), with its extended stem loop structure, interacts with great affinity to both eIF4E and eIF4G, and both eIF4F and its isoform eIFiso4F (4). In contrast, eIF4G alone is necessary and sufficient to support BYDV TE-mediated translation (Treder et al., submitted). eIF4E alone has no affinity for BTE but confers enhancement of eIF4F activity when in complex with eIF4G. On the other hand, the Pea enation mosaic virus, which is
predicted to fold into a T-shape structure, binds eIF4E but requires the all eIF4F complex for translation (Wang Z., personal communication).
This leads to the following questions: (i) What sequence, structure and/or tertiary folding of the core element of a translation element are essential for factor recognition? Has the BTE evolved into a natural RNA aptamer for factors? (ii) What are the roles of the surrounding structures and sequences? Are they required for maintenance of the core element and its protrusion out of the genome for easy accessibility, as indicated by the presence of the long stem holding the 4-way junction (Pettit Kneller, unpublished data)? And (iii) Is sequence specificity of the 5’-3’ base pairing a result of factor recognition and/or configuration.
Regulation of viral translation: I have presented evidence for a novel mechanism of RNA-mediated regulation of gene expression (Chapter 3). The accumulation of an apparently non-translatable sgRNA2 later in infection controls the efficiency of expression of the other viral mRNAs and potentially host cellular translation. The biological relevance of such fine-tuned RNA-mediated mechanism of translation repression as the switch from early expression of the replication complex from the gRNA to favor late expression of the structural proteins from sgRNA1 is clear. An imbalance in the ratio of non-structural versus structural proteins could prevent successful viral infection, by generating insufficient packaging material, too many naked viral genomes, etc. Left unanswered is how a mutant virus, defective of sgRNA2 is still fully competent in replication in protoplasts (8) and in plants (Jackie Jackson, unpublished data). Accumulating data reveal that this mutant virion is fully transmissible by aphids and shows similar infectivity in plants as wild type through a
series of passages (Jackie Jackson, unpublished data). In view of evolutionary pressure on the virus, one would assume that the functional importance of sgRNA2 should correlate with the energy required to produce it. This is relevant also to the highly abundant production of the non-coding sgRNA3, whose function remains unknown.
A new era of research revolves around the fate of translationally repressed RNAs (5,6). These studies address the possible ways in which small non-coding RNAs repress the expression of their target RNAs, either at the level of RNA turnover or translation, and the reversible sequestrations of the mRNAs into specific structures, rich in RNA degrading enzyme, called processing bodies or P-bodies (7). Many interesting questions remain: (i) what is the stage of translation affected by sgRNA2 trans-inhibition (potentially at the initiation level due to a depletion of the translation factors)? and (ii) are viral mRNAs sequestered in similar specific compartiments or bodies in the cytoplasm to go through the translation/replication/packaging cycle? Can the viral mRNAs be directed into those foci to mediate effective repression of viral translation, clearing the RNA of the ribosomes and turning the genomic RNA into replication competent template and/or ready for viral packaging (5). What protects the transcripts from degradation?
The work presented here has explored and solved many truly fascinating aspects of unconventional translational control in the model virus BYDV. It has also uncovered many exciting new questions that will undoubtedly continue to enthrall investigators in our own little corner of the scientific world.