Polarity is an important prerequisite for cell differentiation. The development of cellular asymmetry and cell polarity can be achieved by either selectively transporting key intracellular proteins or vectorial trafficking mRNAs to a subcellular location for local translation. The “A2RE-mRNA trafficking pathway” fits in the latter category, renowned for moving the myelin basic protein (MBP) RNA molecules from the nucleus to cytoplasmic myelin compartment of oligodendrocytes. This prominent pathway is named after an 11-nucleotide cis-acting sequence of “A2 response element (A2RE)”, which is necessary and sufficient for mRNA localization. The universally expressed protein hnRNP A2 was the first identified trans-acting factor bound to the A2RE cis-element directing the A2RE-containing transcripts to their subcellular locations along cytoskeletal components.
In addition to hnRNP A2, hnRNP A3 is the second abundant proteins pulled down by the A2RE-sequence. At the beginning of this project, hnRNP A3 had just been identified as a novel key component along with other A2RE-binding proteins. A lot of structural and functional questions arising from the discovery of hnRNP A3 in the A2RE-context await answers.
The detailed structural knowledge of hnRNP A3 among the A2RE-binding proteins was essential prior to the investigation of its biological involvement in the A2RE-trafficking pathway. Therefore, the overall aim of this project was to characterize the structure of hnRNP A3 isoforms which were purified by binding to the A2RE-sequence. At the same time, the structural comparison with other A2RE-binding proteins was also carried out, aiming to find out any similar or distinct features between these proteins when they were associated together with the same A2RE-sequence. The investigations in this project were established from aspects of genomics, proteomics and cellular location to characterize the hnRNP A3 isoforms. The experimental data, presented in this thesis, were collected reproducibly and comprise all above aspects.
From the genomic aspect, the two splicing forms of hnRNP A3 in rat brain were exclusively identified through the extensive PCR screening. At the protein level, the masses of the intact A2RE-binding proteins were measured by liquid chromatography mass spectrometry (LC/MS) and analyzed. Next, the rat brain A2RE-binding proteins were successfully separated by two-dimensional gel electrophoresis (2-DE), showing a more complicated profile of hnRNP A/B isoforms in the A2RE-context. Each binding components were identified by mass spectrometry (MS) based peptide mass fingerprinting.
Targeting the complexity of hnRNP A3 inside the A2RE-proteome, the post-translational modifications (PTMs) of hnRNP A3 were investigated. Phospho-proteins of the A2RE proteome were detected by blotting and staining. Moreover, the pattern of protein phosphorylation was observed to be isoform-specific between the alternatively spliced isoform pairs. In terms of Arg modifications, the methylation of A3 was confirmed, and six asymmetric dimethylated residues of A3 were successfully identified in the protein Gly-rich region by the bottom-up tandem MS proteomics. The same MS/MS proteomic method also invalidated the occurrence of citrullinated Arg residues in A3.
In the developmental study, the uneven levels between two alternatively spliced HNRPA3 transcripts were discovered. From the observation, the truncated A3b transcripts always maintain a stable higher level than the full-length A3a transcripts. The subcellular distribution patterns of the A3 isoforms were also explored by transfecting GFP-tagged A3 vectors into neuronal cells, in comparison with its endogenous cellular pattern detected by the specific immunostaining method.
In summary, the investigations of hnRNP A3 in this project expanded from its encoding gene to its final cellular location. With more results acquired, the hnRNP A3 isoforms became more significant along with the other key A2RE-binding components A1 and A2/B1. The clarified 2-D profile of the A2RE proteome and unambiguously identified methylated residues of A3 provide insight into the functionally interweaved hnRNP A/B paralogs. Although the conclusions deduced from this project only represent the hnRNP A3 isoforms inside the A2RE-context, the characterization of A3 isoforms under such specific circumstance still provide valuable information for studies of hnRNP A/B paralogs in other fields, opening up a window for studying downstream mechanisms of mRNA biogenesis and metabolism.