Browsing by Author "Been, Michael D"

HDV antigenomic RNA is processed in two distinct pathways; it can be cleaved at the polyA site and polyadenylated to become mRNA for the delta antigens, or the RNA can be cleaved by the antigenomic ribozyme to become full-length antigenomic RNA that is used for synthesis of genomic HDV RNA. The polyA site is located just 33 nucleotides upstream of the ribozyme cleavage site. If processing occurs primarily at the upstream polyA site, there may not be enough full-length antigenomic RNA to support replication. On the other hand, ribozyme cleavage downstream of the polyA site could inhibit polyadenylation by interfering with polyadenylation complex assembly. Thus, it appears that HDV may need a mechanism to control RNA processing so that both products can be generated in the proper amounts during the infection cycle.

A model has been proposed in which the choice between ribozyme cleavage and polyadenylation is determined by alternative RNA secondary structures formed by the polyA sequence (Wadkins and Been 2002). One of the hypothetical structures, AltP2, is a pairing between part of the upstream polyA sequence and the 3' end of the ribozyme sequence. For this model, the same upstream sequence that forms AltP2 could also form a stem loop, P(-1), within the leader, by pairing with sequences located farther upstream. A processing choice is possible because AltP2 is predicted to inhibit ribozyme cleavage and favor polyadenylation resulting in mRNA production, whereas P(-1) would inhibit polyadenylation and favor ribozyme cleavage resulting in full-length replication product.

The P(-1) vs. AltP2 model was tested using an antigenomic HDV ribozyme construct with the 60-nucleotide sequence upstream of the ribozyme cleavage site. This leader sequence contains the proposed polyA sequence elements. In vitro analysis of this construct revealed that the kinetic profile of ribozyme self-cleavage was altered in two ways. Relative to the ribozyme without upstream sequences, the fraction of precursor RNA that cleaved decreased to about 50%, but the active ribozyme fraction cleaved faster. Native gel electrophoresis revealed that the active and inactive precursor RNAs adopted persistent alternative structures, and structure mapping with Ribonuclease T1 and RNase H provided evidence for structures resembling P(-1) and AltP2.

Sequence changes in the 5' leader designed to alter the relative stability of P(-1) and AltP2 increased or decreased the extent of ribozyme cleavage in a predictable way, but disrupting AltP2 did not completely restore ribozyme activity. The analysis of deletion and base change variants supported a second alternative pairing, AltP4, formed by the pyrimidine-rich sequence immediately 5' of the ribozyme cleavage site and a purine-rich sequence from the 5' side of P4. A similar approach was used to test if the effect of disrupting both AltP2 and AltP4 might be additive, and the results suggested that ribozyme precursors with 5' leader sequences could fold into multiple inactive conformations, which can include, but may not be limited to, AltP2, AltP4, or a combination of both.

Luciferase expression constructs with HDV polyA and ribozyme sequences were used to investigate the effects of RNA structure and ribozyme cleavage on polyadenylation in cells. One hypothesis was that P(-1) could inhibit polyadenylation by making the polyA sequence elements less accessible to polyA factors, but sequence changes designed to alter the stability of the stem loop had no effect on polyadenylation. The model also predicts that the ribozyme sequence downstream of the polyA site could affect polyadenylation, possibly in two different ways. Ribozyme cleavage could interfere with polyadenylation by uncoupling transcription from processing, however, the ribozyme sequence might also influence polyadenylation in a manner independent of the ribozyme cleavage activity. As such, the AltP2 structure could potentially have a positive effect on polyadenylation either by inhibiting ribozyme cleavage or by making the polyA signal sequences more accessible to the polyA factors. To distinguish between the effects of ribozyme cleavage and alternative RNA structures, luciferase expression levels from constructs with an HDV polyA sequence followed by the active wild-type ribozyme or the inactive C76u version of the ribozyme were compared. For the wild-type HDV polyA sequence, the active ribozyme reduced expression, whereas the inactive ribozyme control had no effect on expression. However, for the modified leader sequences, which were efficiently polyadenylated in the absence of ribozyme, there were changes in expression that appeared to be independent of ribozyme cleavage. Based on these findings, two alternative models are proposed. One model predicts that protein factors might affect antigenomic RNA processing, and the other model suggests that additional alternative structures, such as AltP4, might influence the choice between ribozyme cleavage and polyadenylation.

Interleukin 7 receptor, IL7R, is expressed exclusively on cells of the lymphoid lineage and its expression is crucial for development and maintenance of T cells. While transcriptional regulation of IL7R expression has been widely studied, its posttranscriptional regulation has only recently been uncovered. Alternative splicing of IL7R exon 6, the only exon that encodes the transmembrane domain of the receptor, results in membrane-bound (exon 6 included) and soluble (exon 6 skipped) IL7R isoforms, respectively. Interestingly, the inclusion of exon 6 is affected by a single-nucleotide polymorphism associated with the risk of developing multiple sclerosis, a prototypic demyelinating disease of the central nervous system. Given the potential association of exon 6 inclusion with multiple sclerosis, we investigated the cis-acting elements and trans-acting factors that regulate exon 6 splicing.

We utilized mutagenesis of exon 6 and surrounding introns to identify multiple exonic and intronic cis-acting regulatory elements that impact inclusion of exon 6. At least two of these elements, one exonic splicing silencer and one exonic splicing enhancer, are located in the direct vicinity of the MS-associated SNP. We also uncovered a consensus polyadenylation signal, AAUAAA in intron 6 of IL7R, 16 nucleotides downstream from exon 6 5' splice site, and showed that mutations to this site resulted in an increase in exon 6 inclusion. Additionally, we determined that the 5' splice site of exon 6 is weak. We propose that this site may be responsible for exon 6 splicing regulation.

Using tobramycin RNA affinity chromatography followed by mass spectrometry, we identified trans-acting protein factors that bind exon 6 and regulate its splicing. These experiments identified cleavage and polyadenylation specificity factor 1 (CPSF1) among protein binding candidates. siRNA-mediated knockdown of CPSF1 resulted in an increase in exon 6 inclusion, consistent with the results of mutations to the CPSF1 binding site. Correspondingly, CPSF1 depletion had no effect on a minigene with a mutation in the intronic polyadenylation site. Finally, 3'RACE and RT-PCR experiments on RNA from Jurkat cells suggested that the intronic AAUAAA site is utilized at low frequency by the polyadenylation machinery to produce a novel polyadenylated mRNA isoform. Together, our results suggest that competing pre-mRNA splicing and polyadenylation may regulate exon 6 inclusion and resultant levels of functional IL7R produced. Since the intronic polyadenylated isoform of IL7R is predicted to be translated into a membrane-bound protein product with a shortened, signal transduction-incompetent cytoplasmic tail, this may be relevant for both T cell biology and development of multiple sclerosis.