Characterisation of the mouse hnRNP A2/B1 gene and its protein isoforms

Hatfield, Jodie (2007). Characterisation of the mouse hnRNP A2/B1 gene and its protein isoforms PhD Thesis, School of Molecular and Microbial Sciences, University of Queensland.

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Author Hatfield, Jodie
Thesis Title Characterisation of the mouse hnRNP A2/B1 gene and its protein isoforms
School, Centre or Institute School of Molecular and Microbial Sciences
Institution University of Queensland
Publication date 2007
Thesis type PhD Thesis
Supervisor Professor Ross Smith
Abstract/Summary The heterogeneous nuclear ribonucleoprotein (hnRNP) A2/B1 gene participates in at least six cellular processes: telomere biogenesis and maintenance, packaging of nascent premRNAs, alternative splicing, mRNA stability, mRNA cytoplasmic trafficking and translation initiation. At the start of this project the mouse gene had not yet been described, however, the human gene was known to produce two protein isoforms, A2 and B1, but the functional boundaries between these two proteins were ill defined. The characterisation of the mouse hnRNP A2/B1 gene and its protein isoforms was, therefore, undertaken to facilitate research in mouse which would help to define functional differences between the protein isoforms. The mouse gene was found to be a 14 kb, single-copy gene located on chromosome 6 B3, comprised of 12 exons and 11 introns. This gene was regulated by a housekeeping-style promoter which was predicted to be affected by processes such as cell differentiation and maturation, cell cycle progression and stress responses. Transcription from this gene, and the human orthologue, gave rise to a number of RNAs that encoded four protein isoforms through alternative splicing of exons 2 and 9: B1, A2, B1b and A2b. In mouse, the expression and alternative splicing of the hnRNP A2/B1 transcripts were influenced by tissue-type and age. A subset of these transcripts also exhibited alterations to their 3’ UTRs such as retention of the 11th intron, alternative polyadenylation and 3’ UTR extension. The extended 3’ UTR region, intron 11 and sequence flanking the alternatively spliced exons were highly conserved between mouse and human (over 90%) and were predicted to contain regulatory motifs that governed post-transcriptional modifications to the RNA messages. The mouse genome was also shown to contain at least six processed pseudogenes derived from the hnRNP A2/B1 gene, five of which were unlikely to be functional due to frequent variations from the real gene sequence, including insertions, deletions and rearrangements. The remaining pseudogene, pseudogene 1, could give rise to an independent source of A2 proteins and its status is yet to be determined. To date, it is unclear what role each of the four hnRNP A2/B1 protein isoforms play in the cell. It was proposed that protein isoforms with a unique functional role would exhibit differences in subcellular localisation and expression patterns. In order to test this hypothesis, a number of biological tools were developed to distinguish between the highly similar proteins. In addition to two rabbit polyclonal antibodies previously raised in our lab that recognised all four protein isoforms (HA2) and isoforms B1 and B1b (HB1), another four polyclonal antibodies were raised in rabbits and chickens. These antibodies were raised to recognise the following combinations: all four proteins (chicken HA2), A2 and A2b (chicken Hx1/3), B1 and A2 (rabbit Hx9) and A2b and B1b (rabbit Hx8/10). These antibodies were demonstrated to be suitable for Western analyses and immunostaining studies, although antibodies Hx1/3, Hx8/10 and chicken HA2 will require further purification to reduce nonspecific signals seen in immunostaining experiments. A number of fluorescently tagged fusion proteins were also engineered for each of the A2/B1 protein isoforms. Western analyses showed that in mouse tissues and a number of immortalised cell lines, A2 and B1 were the major hnRNP A-type proteins present. Their abundance varied between tissue- and cell-type, but both were constitutively expressed. In contrast, A2b was only present in younger mouse tissues and was absent from most immortalised cell lines, while B1b was not detected in the samples examined. Localisation studies showed that in the immortalised cell lines, HaCaT and PC12, the expressed protein isoforms and related fusion proteins localised to the cell nucleus but were excluded from the nucleoli and exhibited distinct perinucleolar staining. Little difference was observed between the localisation patterns of the protein isoforms A2 and B1 in these cell types. Interestingly in oligodendrocytes, a cell type known to carry out A2-dependent mRNA trafficking, B1 was localised to the nucleus, A2 was predominantly nuclear but also exhibited some cytoplasmic localisation and A2b was distributed almost evenly between the nucleus and cytoplasm (M. Maggipinto and R. Smith, personal communication). Hence, differences were observed in the localisation of these proteins but were cell-type dependent. In conclusion, the hnRNP A2/B1 splice variants and their protein products were differentially expressed temporally and spatially, suggesting that these highly similar protein isoforms were likely to possess unique functional roles. Future research using the biological tools developed and described in this thesis, will be directed towards further defining the different roles each of the protein isoforms may have in the cellular processes associated with the hnRNP A2/B1 gene. Lastly, the hnRNP A2/B1 gene belongs to a sub-family of hnRNP genes that have arisen through chromosomal duplication events and include hnRNPs A1 and A3. Comparisons of their gene structures and subsequent RT-PCR assays have revealed that, in addition to the two previously described splice variants of the hnRNP A3 gene, A3a and A3b, the eighth exon of the A3 transcript was also alternatively spliced in humans and gave rise to two new variants termed A3c and A3d.

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