Polyadenylation in rice tungro bacilliform virus: cis-acting signals and regulation.

The polyadenylation signal of rice tungro bacilliform virus (RTBV) was characterized by mutational and deletion analysis. The cis-acting signals required to direct polyadenylation conformed to what is known for plant poly(A) signals in general and were very similar to those of the related cauliflower mosaic virus. Processing was directed by a canonical AAUAAA poly(A) signal, an upstream UG-rich region considerably enhanced processing efficiency, and sequences downstream of the cleavage site were not required. When present at the end of a transcription unit, the cis-acting signals for 3'-end processing were highly efficient in both monocot (rice) and dicot (Nicotiana plumbaginifolia) protoplasts. In a promoter-proximal position, as in the viral genome, the signal was also efficiently processed in rice protoplasts, giving rise to an abundant "short-stop" (SS-) RNA. The proportion of SS-RNA was considerably lower in N. plumbaginifolia protoplasts. In infected plants, SS-RNA was hardly detectable, suggesting either that SS-RNA is unstable in infected plants or that read-through of the promoter-proximal poly(A) site is very efficient. SS-RNA is readily detectable in transgenic rice plants (A. Klöti, C. Henrich, S. Bieri, X. He, G. Chen, P. K. Burkhardt, J. Wünn, P. Lucca, T. Hohn, I. Potrylus, and J. Fütterer, 1999. Plant Mol. Biol. 40:249-266), thus the absence of SS-RNA in infected plants can be attributed to poly(A) site bypass in the viral context to ensure production of the full-length pregenomic viral RNA. RTBV poly(A) site suppression thus depends both on context and the expression system; our results suggest that the circular viral minichromosome directs assembly of a transcription-processing complex with specific properties to effect read-through of the promoter-proximal poly(A) signal.

Rice tungro bacilliform virus open reading frames II and III are translated from polycistronic pregenomic RNA by leaky scanning.

Posttranscriptional components of the gene expression mechanism of rice tungro bacilliform virus (RTBV) were studied in transiently transfected protoplasts. RTBV translates several open reading frames from a polycistronic mRNA by leaky scanning. This mechanism is supported by the particular sequence features of the corresponding genome region and does not require a virus-encoded transactivator.

Efficient transcription from the rice tungro bacilliform virus promoter requires elements downstream of the transcription start site.

Elements downstream of the transcription start site enhance the activity of the rice tungro bacilliform virus (RTBV) promoter in protoplasts derived from cultured rice cells. This enhancer region was located to the first 90 nucleotides of the RTBV leader sequence. Within this region, at least two components which act together to enhance expression from the RTBV promoter could be identified. One is a position- and orientation-independent DNA element within a CT-rich region, and the other is a position-dependent element. Either element was found to be capable of acting independently on a heterologous promoter. The enhancer activity of the DNA element correlates with specific binding of nuclear proteins. Nuclear proteins also recognize an RNA transcript covering the first 90 nucleotides of the RTBV leader.

Plant mRNA 3'-end formation.

Our understanding of how the 3' ends of mRNAs are formed in plants is rudimentary compared to what we know about this process in other eukaryotes. The salient features of plant pre-mRNAs that signal cleavage and polyadenylation remain obscure, and the biochemical mechanism is as yet wholly uncharacterized. Nevertheless, despite the lack of universally conserved cis-acting motifs, a common underlying architecture is emerging from functional analyses of plant poly(A) signals, allowing meaningful comparison with components of poly(A) signals in other eukaryotes. A plant poly(A) signal consists of one or more near-upstream elements (NUE), each directing processing at a poly(A) site a short distance downstream of it, and an extensive far-upstream element (FUE) that enhances processing efficiency at all sites. By analogy with other systems, a model for a plant 3'-end processing complex can be proposed. Plant poly(A) polymerases have been isolated and partially characterised. These, together with hints that some processing factors are conserved in different organisms, opens promising avenues toward initial characterisation of the trans-acting factors involved in 3'-end formation of mRNAs in higher plants.

Molecular characterization of the spliceosomal proteins U1A and U2B" from higher plants.

In addition to their role in pre-mRNA splicing, the human spliceosomal proteins U1A and U2B" are important models of how RNP motif-containing proteins execute sequence-specific RNA binding. Genes encoding U1A and U2B" have been isolated from potato and thereby provide the only evolutionary comparison available for both proteins and represent the only full-length genes encoding plant spliceosomal proteins to have been cloned and characterized. In vitro RNA binding experiments revealed the ability of potato U2B" to interact with human U2A' to enhance sequence-specific binding and to distinguish cognate RNAs of either plant or animal origin. A comparison of the sequence of U1A and U2B" proteins indicated that multiple residues which could affect RNP motif conformation probably govern the specific distinction in RNA binding by these proteins. Since human U1A modulates polyadenylation in vertebrates, the possibility that plant U1A might be exploited in the characterization of this process in plants was examined. However, unlike vertebrate U1A, neither U1A from potato nor Arabidopsis bound their own mRNA and no evidence for binding to upstream efficiency elements in polyadenylation signals was obtained, suggesting that plant U1A is not involved in polyadenylation.

The contribution of AAUAAA and the upstream element UUUGUA to the efficiency of mRNA 3'-end formation in plants.

The requirement for sequence specificity in the AAUAAA motif of the cauliflower mosaic virus (CaMV) polyadenylation signal was examined by saturation mutagenesis. While deletion of AAUAAA almost abolished processing at the CaMV polyadenylation site, none of the 18 possible single base mutations had a dramatic effect on processing efficiency. The effect of replacing all six nucleotides simultaneously varied depending on the sequence used, but some replacements were as detrimental as the deletion mutant. Taken together, these results confirm that AAUAAA is an essential component of the CaMV polyadenylation signal, but indicate that a high degree of sequence variation can be tolerated. A repeated UUUGUA motif was identified as an important upstream accessory element of the CaMV polyadenylation signal. This sequence was able to induce processing at a heterologous polyadenylation site in a sequence-specific and additive manner. The effect of altering the spacing between this upstream element and the AAUAAA was examined; moving these two elements closer together or further apart reduces the processing efficiency. The upstream element does not function to signal processing at the CaMV polyadenylation site if placed downstream of the cleavage site. Analysis of further upstream sequences revealed that almost all of the 200 nt fragment required for maximal processing contributes positively to processing efficiency. Furthermore, isolated far upstream sequences distinct from UUUGUA were also able to induce processing at a heterologous polyadenylation site.

Retrotransposon-like nature of Tp1 elements: implications for the organisation of highly repetitive, hypermethylated DNA in the genome of Physarum polycephalum.

The repetitive fraction of the genome of the eukaryotic slime mould Physarum polycephalum is dominated by the Tp1 family of highly repetitive retrotransposon-like sequences. Tp1 elements consist of two terminal direct repeats of 277bp which flank an internal domain of 8.3kb. They are the major sequence component in the hypermethylated (M+) fraction of the genome where they have been found exclusively in scrambled clusters of up to 50kb long. Scrambling is thought to have arisen by insertion of Tp1 into further copies of the same sequence. In the present study, sequence analysis of cloned Tp1 elements has revealed striking homologies of the predicted amino acid sequence to several highly conserved domains characteristic of retrotransposons. The relative order of the predicted coding regions indicates that Tp1 elements are more closely related to copia and Ty than to retroviruses. Self-integration and methylation of Tp1 elements may function to limit transposition frequency. Such mechanisms provide a possible explanation for the origin and organisation of M + DNA in the Physarum genome.

Identification of a second retrotransposon-related element in the genome of Physarum polycephalum.

The repetitive fraction of the genome of the eukaryotic slime mould Physarum polycephalum is dominated by Tp1, a family of retrotransposon-like sequences. Tp1 elements are arranged in scrambled clusters probably arising from integration of the element into copies of its own sequence. The present report describes a second sequence family, Tp2, which has been identified within cloned DNA segments of scrambled Tp1 sequences. Like Tp1, the Tp2 element is structurally related to retrotransposons, having long terminal direct repeats and being flanked by an apparent target site duplication, but its relatively short length (1.68 kb) indicates that it is probably incapable of encoding all the functions necessary for its own mobilisation. Analysis of the coding potential of the Tp2 element supports this view, although a striking homology to a nucleic acid binding domain common to many retrotransposons was identified. As with Tp1, putative regulatory signals can be identified in the LTRs of Tp2. Identical arrangements of Tp2 with respect to Tp1 in more than one independently derived clone indicate that non-functional copies of Tp2 may be mobilised as part of a Tp1 transcriptional unit.