Translon: a single term for translated regions.
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Letter↗
Biomedical subjects
W Allen Miller
Publications and source records attributed to W Allen Miller.
Dynamics of a mobile RNA of potato involved in a long-distance signaling pathway.
BEL1-like transcription factors interact with Knotted1 types to regulate numerous developmental processes. In potato (Solanum tuberosum), the BEL1 transcription factor St BEL5 and its protein partner POTH1 regulate tuber formation by mediating hormone levels in the stolon tip. The accumulation of St BEL5 RNA increases in response to short-day photoperiods, inductive for tuber formation. RNA detection methods and heterografting experiments demonstrate that BEL5 transcripts are present in phloem cells and move across a graft union to localize in stolon tips, the site of tuber induction. This movement of RNA to stolon tips is correlated with enhanced tuber production. Overexpression of BEL5 transcripts that include the untranslated sequences of the BEL5 transcript endows transgenic lines with the capacity to overcome the inhibitory effects of long days on tuber formation. Addition of the untranslated regions leads to preferential accumulation of the BEL5 RNA in stolon tips under short-day conditions. Using a leaf-specific promoter, the movement of BEL5 RNA to stolon tips was facilitated by a short-day photoperiod, and this movement was correlated with enhanced tuber production. These results implicate the transcripts of St BEL5 in a long-distance signaling pathway that are delivered to the target organ via the phloem stream.
Gene Expression Regulation, Plant↗
A glassy-winged sharpshooter cell line supports replication of Rhopalosiphum padi virus (Dicistroviridae).
Rhopalosiphum padi virus (RhPV) (family Dicistroviridae; genus Cripavirus) is an icosahedral aphid virus with a 10kb positive-sense RNA genome. To study the molecular biology of RhPV, identification of a cell line that supports replication of the virus is essential. We screened nine cell lines derived from species within the Lepidoptera, Diptera and Hemiptera for susceptibility to RhPV following RNA transfection. We observed cytopathic effects (CPE) only in cell lines derived from hemipterans, specifically GWSS-Z10 cells derived from the glassy winged sharp shooter, Homalodisca coagulata and DMII-AM cells derived from the corn leaf hopper, Dalbulus maidis. Translation and appropriate processing of viral gene products, RNA replication and packaging of virus particles in the cytoplasm of GWSS-Z10 cells were examined by Western blot analysis, Northern blot hybridization and electron microscopy. Infectivity of the GWSS-Z10 cell derived-virus particles to the bird cherry-oat aphid, R. padi, was confirmed by RT-PCR and Western blot. The GWSS-Z10 cell line provides a valuable tool to investigate replication, structure and assembly of RhPV.
Animals↗
Structures required for poly(A) tail-independent translation overlap with, but are distinct from, cap-independent translation and RNA replication signals at the 3' end of Tobacco necrosis virus RNA.
Tobacco necrosis necrovirus (TNV) RNA lacks both a 5' cap and a poly(A) tail but is translated efficiently, owing in part to a Barley yellow dwarf virus (BYDV)-like cap-independent translation element (BTE) in its 3' untranslated region (UTR). Here, we identify sequence downstream of the BTE that is necessary for poly(A) tail-independent translation in vivo by using RNA encoding a luciferase reporter gene flanked by viral UTRs. Deletions and point mutations caused loss of translation that was restored by adding a poly(A) tail, and not by adding a 5' cap. The two 3'-proximal stem-loops in the viral genome contribute to poly(A) tail-independent translation, as well as RNA replication. For all necroviruses, we predict a conserved 3' UTR secondary structure that includes the BTE at one end of a long helical axis and the stem-loops required for poly(A) tail-independent translation and RNA replication at the other end. This work shows that a viral genome can harbor distinct cap- and poly(A) tail-mimic sequences in the 3' UTR.
3' Untranslated Regions↗
Oscillating kissing stem-loop interactions mediate 5' scanning-dependent translation by a viral 3'-cap-independent translation element.
The 3'-untranslated regions (UTRs) of a group of novel uncapped viral RNAs allow efficient translation initiation at the 5'-proximal AUG. A well-characterized model is the Barley yellow dwarf virus class of cap-independent translation elements (BTE). It facilitates translation by forming kissing stem-loops between the BTE in the 3'-UTR and a BTE-complementary loop in the 5'-UTR. Here we investigate the mechanisms of the long-distance interaction and ribosome entry on the RNA. Upstream AUGs or 5'-extensions of the 5'-UTR inhibit translation, indicating that, unlike internal ribosome entry sites in many viral RNAs, the BTE relies on 5'-end-dependent ribosome scanning. Cap-independent translation occurs when the kissing sites are moved to different regions in either UTR, including outside of the BTE. The BTE can even confer cap-independent translation when fused to the 3'-UTR of a reporter RNA harboring dengue virus sequences that cause base-pairing between the 3'- and 5'-ends. Thus, the BTE serves as a functional sensor to detect sequences capable of long-distance base-pairing. We propose that the kissing interaction is repeatedly disrupted by the scanning ribosome and re-formed in an oscillating process that regulates ribosome entry on the RNA.
3' Untranslated Regions↗
Translational control in positive strand RNA plant viruses.
The great variety of genome organizations means that most plant positive strand viral RNAs differ from the standard 5'-cap/3'-poly(A) structure of eukaryotic mRNAs. The cap and poly(A) tail recruit initiation factors that support the formation of a closed loop mRNA conformation, the state in which translation initiation is most efficient. We review the diverse array of cis-acting sequences present in viral mRNAs that compensate for the absence of a cap, poly(A) tail, or both. We also discuss the cis-acting sequences that control translation strategies that both amplify the coding potential of a genome and regulate the accumulations of viral gene products. Such strategies include leaky scanning initiation of translation of overlapping open reading frames, stop codon readthrough, and ribosomal frameshifting. Finally, future directions for research on the translation of plant positive strand viruses are discussed.
3' Untranslated Regions↗
trans regulation of cap-independent translation by a viral subgenomic RNA.
Many positive-strand RNA viruses generate 3'-coterminal subgenomic mRNAs to allow translation of 5'-distal open reading frames. It is unclear how viral genomic and subgenomic mRNAs compete with each other for the cellular translation machinery. Translation of the uncapped Barley yellow dwarf virus genomic RNA (gRNA) and subgenomic RNA1 (sgRNA1) is driven by the powerful cap-independent translation element (BTE) in their 3' untranslated regions (UTRs). The BTE forms a kissing stem-loop interaction with the 5' UTR to mediate translation initiation at the 5' end. Here, using reporter mRNAs that mimic gRNA and sgRNA1, we show that the abundant sgRNA2 inhibits translation of gRNA, but not sgRNA1, in vitro and in vivo. This trans inhibition requires the functional BTE in the 5' UTR of sgRNA2, but no translation of sgRNA2 itself is detectable. The efficiency of translation of the viral mRNAs in the presence of sgRNA2 is determined by proximity to the mRNA 5' end of the stem-loop that kisses the 3' BTE. Thus, the gRNA and sgRNA1 have "tuned" their expression efficiencies via the site in the 5' UTR to which the 3' BTE base pairs. We conclude that sgRNA2 is a riboregulator that switches off translation of replication genes from gRNA while permitting translation of structural genes from sgRNA1. These results reveal (i) a new level of control of subgenomic-RNA gene expression, (ii) a new role for a viral subgenomic RNA, and (iii) a new mechanism for RNA-mediated regulation of translation.
3' Untranslated Regions↗
Long-distance RNA-RNA interactions in plant virus gene expression and replication.
The vast majority of plant and animal viruses have RNA genomes. Viral gene expression and replication are controlled by cis-acting elements in the viral genome, which have been viewed conventionally as localized structures. However, recent research has altered this perception and provided compelling evidence for cooperative activity involving distantly positioned RNA elements. This chapter focuses on viral RNA elements that interact across hundreds or thousands of intervening nucleotides to control translation, genomic RNA synthesis, and subgenomic mRNA transcription. We discuss evidence supporting the existence and function of the interactions, and speculate on the regulatory roles that such long-distance interactions play in the virus life cycle. We emphasize viruses in the Tombusviridae and Luteoviridae families in which long-distance interactions are best characterized, but similar phenomena in other viruses are also discussed. Many more examples likely remain undiscovered.
Gene Expression Regulation, Viral↗
Cap-independent translation of plant viral RNAs.
The RNAs of many plant viruses lack a 5' cap and must be translated by a cap-independent mechanism. Here, we discuss the remarkably diverse cap-independent translation elements that have been identified in members of the Potyviridae, Luteoviridae, and Tombusviridae families, and genus Tobamovirus. Many other plant viruses have uncapped RNAs but their translation control elements are uncharacterized. Cap-independent translation elements of plant viruses differ strikingly from those of animal viruses: they are smaller (<200 nt), some are located in the 3' untranslated region, some require ribosome scanning from the 5' end of the mRNA, and the 5' UTR elements are much less structured than those of animal viruses. We discuss how these elements may interact with host translation factors, and speculate on their mechanism of action and their roles in the virus replication cycle. Much remains to be learned about how these elements enable plant viruses to usurp the host translational machinery.
Base Sequence↗
A simple wax-embedding method for isolation of aphid hemolymph for detection of luteoviruses in the hemocoel.
A protocol for isolating hemolymph from viruliferous aphids has been developed. This method uses warm melted wax to immobilize the aphid. Following removal of a hind leg, the hemolymph can be collected readily. Flushing with RNase-free water allows for collection of sufficient hemolymph for RNA extraction from individual aphids. The extracted RNA was successfully used for detection of barley yellow dwarf virus (BYDV) and pea enation mosaic virus (PEMV) from individual viruliferous Rhopalosiphum padi and Acyrthosiphon pisum aphids, respectively. A TaqMan real-time RT-PCR protocol for quantitation of PEMV in the hemolymph of individual aphids was developed. The wax-embedding hemolymph collection technique provides a useful tool for studying molecular interactions between persistent and circulative plant viruses and their insect vectors.
Animals↗
Subgenomic RNA as a riboregulator: negative regulation of RNA replication by Barley yellow dwarf virus subgenomic RNA 2.
Barley yellow dwarf virus (BYDV) generates three 3'-coterminal subgenomic RNAs (sgRNAs) in infected cells. Translation of BYDV genomic RNA (gRNA) and sgRNA1 is mediated by the BYDV cap-independent translation element (BTE) in the 3' untranslated region. sgRNAs 2 and 3 are unlikely to be mRNAs. We proposed that accumulation of sgRNA2, which contains the BTE in its 5' UTR, regulates BYDV replication by trans-inhibiting translation of the viral polymerase from genomic RNA (gRNA). Here, we tested this hypothesis and found that: (i) co-inoculation of the BTE or sgRNA2 with BYDV RNA inhibits BYDV RNA accumulation in protoplasts; (ii) Brome mosaic virus (BMV), engineered to contain the BTE, trans-inhibits BYDV replication; and (iii) sgRNA2 generated during BYDV infection trans-inhibits both GFP expression from BMV RNA and translation of a non-viral reporter mRNA. We conclude that sgRNA2, via its BTE, functions as a riboregulator to inhibit translation of gRNA. This may make gRNA available as a replicase template and for encapsidation. Thus, BYDV sgRNA2 joins a growing list of trans-acting regulatory RNAs.
3' Untranslated Regions↗
Cis and trans requirements for rolling circle replication of a satellite RNA.
Satellite RNAs usurp the replication machinery of their helper viruses, even though they bear little or no sequence similarity to the helper virus RNA. In Cereal yellow dwarf polerovirus serotype RPV (CYDV-RPV), the 322-nucleotide satellite RNA (satRPV RNA) accumulates to high levels in the presence of the CYDV-RPV helper virus. Rolling circle replication generates multimeric satRPV RNAs that self-cleave via a double-hammerhead ribozyme structure. Alternative folding inhibits formation of a hammerhead in monomeric satRPV RNA. Here we determine helper virus requirements and the effects of mutations and deletions in satRPV RNA on its replication in oat cells. Using in vivo selection of a satRPV RNA pool randomized at specific bases, we found that disruption of the base pairing necessary to form the non-self-cleaving conformation reduced satRPV RNA accumulation. Unlike other satellite RNAs, both the plus and minus strands proved to be equally infectious. Accordingly, very similar essential replication structures were identified in each strand. A different region is required only for encapsidation. The CYDV-RPV RNA-dependent RNA polymerase (open reading frames 1 and 2), when expressed from the nonhelper Barley yellow dwarf luteovirus, was capable of replicating satRPV RNA. Thus, the helper virus's polymerase is the sole determinant of the ability of a virus to replicate a rolling circle satellite RNA. We present a framework for functional domains in satRPV RNA with three types of function: (i) conformational control elements comprising an RNA switch, (ii) self-functional elements (hammerhead ribozymes), and (iii) cis-acting elements that interact with viral proteins.
Avena↗
The 3' untranslated region of tobacco necrosis virus RNA contains a barley yellow dwarf virus-like cap-independent translation element.
RNAs of many viruses are translated efficiently in the absence of a 5' cap structure. The tobacco necrosis virus (TNV) genome is an uncapped, nonpolyadenylated RNA whose translation mechanism has not been well investigated. Computational analysis predicted a cap-independent translation element (TE) within the 3' untranslated region (3' UTR) of TNV RNA that resembles the TE of barley yellow dwarf virus (BYDV), a luteovirus. Here we report that such a TE does indeed exist in the 3' UTR of TNV strain D. Like the BYDV TE, the TNV TE (i) functions both in vitro and in vivo, (ii) requires additional sequence for cap-independent translation in vivo, (iii) has a similar secondary structure and the conserved sequence CGGAUCCUGGGAAACAGG, (iv) is inactivated by a four-base duplication in this conserved sequence, (v) can function in the 5' UTR, and (vi) when located in its natural 3' location, may form long-distance base pairing with the viral 5' UTR that is conserved and probably required. The TNV TE differs from the BYDV TE by having only three helical domains instead of four. Similar structures were found in all members of the Necrovirus genus of the Tombusviridae family, except satellite tobacco necrosis virus, which harbors a different 3' cap-independent translation domain. The presence of the BYDV-like TE in select genera of different families indicates that phylogenetic distribution of TEs does not follow standard viral taxonomic relationships. We propose a new class of cap-independent TE called BYDV-like TE.
3' Untranslated Regions↗
A -1 ribosomal frameshift element that requires base pairing across four kilobases suggests a mechanism of regulating ribosome and replicase traffic on a viral RNA.
Programmed -1 ribosomal frameshifting is necessary for translation of the polymerase genes of many viruses. In addition to the consensus elements in the mRNA around the frameshift site, we found previously that frameshifting on Barley yellow dwarf virus RNA requires viral sequence located four kilobases downstream. By using dual luciferase reporter constructs, we now show that a predicted loop in the far downstream frameshift element must base pair to a bulge in a bulged stem loop adjacent to the frameshift site. Introduction of either two or six base mismatches in either the bulge or the far downstream loop abolished frameshifting, whereas mutations in both sites that restored base pairing reestablished frameshifting. Likewise, disruption of this base pairing abolished viral RNA replication in plant cells, and restoration of base pairing completely reestablished virus replication. We propose a model in which Barley yellow dwarf virus uses this and another long-distance base-pairing event required for cap-independent translation to allow the replicase copying from the 3' end to shut off translation of upstream ORFs and free the RNA of ribosomes to allow unimpeded replication. This would be a means of solving the "problem," common to positive strand RNA viruses, of competition between ribosomes and replicase for the same RNA template.
3' Untranslated Regions↗
The 3prime prime or minute-terminal structure required for replication of Barley yellow dwarf virus RNA contains an embedded 3prime prime or minute end.
We determined the 3prime prime or minute-terminal primary and secondary structures required for replication of Barley yellow dwarf virus (BYDV) RNA in oat protoplasts. Computer predictions, nuclease probing, phylogenetic comparisons, and replication assays of specific mutants and chimeras revealed that the 3prime prime or minute-terminal 109 nucleotides (nt) form a structure with three to four stem-loops followed by a coaxially stacked helix incorporating the last four nt [(A/U)CCC]. Sequences upstream of the 109-nt region also contributed to RNA accumulation. The base-pairing but not the sequences or bulges in the stems were essential for replication, but any changes to the 3prime prime or minute-terminal helix destroyed replication. The two 3prime prime or minute-proximal tetraloops tolerated all changes, but the two 3prime prime or minute-distal tetraloops gave most efficient replication if they fit the GNRA consensus. A mutant lacking the 3prime prime or minute-proximal stem-loop produced elevated levels of less-than-full-length minus strands, and no (+) strand. We propose that a "pocket" structure is the origin of (minus sign)-strand synthesis, which is negatively regulated by the inaccessible conformation of the 3prime prime or minute terminus, thus favoring a high (+)/(minus sign) ratio. This 3prime prime or minute structure and the polymerase homologies suggest that genus Luteovirus is more closely related to the Tombusviridae family than to other Luteoviridae genera.
Avena↗