biology Review hnRNP A/B Proteins: An Encyclopedic Assessment of Their Roles in Homeostasis and Disease Patricia A. Thibault 1,2 , Aravindhan Ganesan 3, Subha Kalyaanamoorthy 4, Joseph-Patrick W. E. Clarke 1,5,6 , Hannah E. Salapa 1,2 and Michael C. Levin 1,2,5,6,* 1 Office of the Saskatchewan Multiple Sclerosis Clinical Research Chair, University of Saskatchewan, Saskatoon, SK S7K 0M7, Canada; [email protected] (P.A.T.); [email protected] (J.-P.W.E.C.); [email protected] (H.E.S.) 2 Department of Medicine, Neurology Division, University of Saskatchewan, Saskatoon, SK S7N 0X8, Canada 3 ArGan’s Lab, School of Pharmacy, Faculty of Science, University of Waterloo, Waterloo, ON N2L 3G1, Canada; [email protected] 4 Department of Chemistry, Faculty of Science, University of Waterloo, Waterloo, ON N2L 3G1, Canada; [email protected] 5 Department of Health Sciences, College of Medicine, University of Saskatchewan, Saskatoon, SK S7N 5E5, Canada 6 Department of Anatomy, Physiology and Pharmacology, University of Saskatchewan, Saskatoon, SK S7N 5E5, Canada * Correspondence: [email protected] Simple Summary: The hnRNP A/B family of proteins (comprised of A1, A2/B1, A3, and A0) contributes to the regulation of the majority of cellular RNAs. Here, we provide a comprehensive overview of what is known of each protein’s functions, highlighting important differences between them. While there is extensive information about A1 and A2/B1, we found that even the basic Citation: Thibault, P.A.; Ganesan, A.; functions of the A0 and A3 proteins have not been well-studied. We also noted that the regulation Kalyaanamoorthy, S.; Clarke, and tissue distribution of all four of the proteins and their different isoforms require further study. J.-P.W.E.; Salapa, H.E.; Levin, M.C. Finally, since these proteins together play such a central role in regulating the cell’s RNA, we call for hnRNP A/B Proteins: An careful comparative examination of these proteins to better define the precise boundaries of each Encyclopedic Assessment of Their Roles in Homeostasis and Disease. protein’s role in cell function and disease. Biology 2021, 10, 712. https:// doi.org/10.3390/biology10080712 Abstract: The hnRNP A/B family of proteins is canonically central to cellular RNA metabolism, but due to their highly conserved nature, the functional differences between hnRNP A1, A2/B1, A0, and Academic Editors: Stylianos A3 are often overlooked. In this review, we explore and identify the shared and disparate homeostatic Ravanidis and Epaminondas Doxakis and disease-related functions of the hnRNP A/B family proteins, highlighting areas where the proteins have not been clearly differentiated. Herein, we provide a comprehensive assembly of Received: 15 June 2021 the literature on these proteins. We find that there are critical gaps in our grasp of A/B proteins’ Accepted: 21 July 2021 alternative splice isoforms, structures, regulation, and tissue and cell-type-specific functions, and Published: 24 July 2021 propose that future mechanistic research integrating multiple A/B proteins will significantly improve our understanding of how this essential protein family contributes to cell homeostasis and disease. Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in Keywords: RNA binding protein; hnRNP A/B; RNA metabolism; splicing; RNA trafficking; RNA published maps and institutional affil- granules; RNA homeostasis; telomeres; neurodegenerative disease iations. 1. A Brief History of the hnRNPs Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. Heterogeneous nuclear ribonucleoproteins, “hnRNPs”, were first defined as proteins This article is an open access article associated with heterogeneous nuclear RNAs (hnRNAs), but not with chromatin, nucle- distributed under the terms and olar RNAs, nor polyribosomal RNAs [1,2]. These “hnRNAs” were later understood to conditions of the Creative Commons be pre-mRNAs and spliced mRNAs that had not yet left the nucleus. Early exploration Attribution (CC BY) license (https:// of hnRNPs was largely chemical and biochemical in nature, with initial studies isolating creativecommons.org/licenses/by/ purified cellular nuclei, and employing sucrose and cesium chloride gradients to isolate 4.0/). protein:RNA complexes that sedimented at approximately 40S. RNase treatments and Biology 2021, 10, 712. https://doi.org/10.3390/biology10080712 https://www.mdpi.com/journal/biology Biology 2021, 10, 712 2 of 40 cutting-edge technologies like UV-crosslinking, two-dimensional gel electrophoresis, and Edman protein sequencing were then employed to define the chemical characteristics and amino acid sequences of the proteins of interest [3–10]. Speculation about protein function was limited since individual hnRNPs’ preference for DNA or RNA binding, and any sequence specificity, was not immediately apparent [2,10–12]. Instead, in electron micrographs, hnRNPs appeared to bundle nuclear RNAs much the same way histones bundle chromosomal DNA [13]. Since these protein complexes appeared to be exclusively found in the nuclear fraction of cells [14,15], and proteins with similar molecular weights and isoelectric points (pI) were occasionally associated with chromatin, they were hypothe- sized to bundle and store newly-transcribed mRNAs until the mRNAs were needed in the cytoplasm [2,9,12]. With increasingly sophisticated biochemical tools, including the advent of monoclonal antibodies [10,16–18], hnRNPs were better defined as approximately twenty-five different nuclear proteins (hnRNP A through U) with varying affinity for hnRNAs [7,19]. Some hnRNPs were separately identified and characterized in other contexts, and are now more commonly known by other names: Auf1 (hnRNP D), PCBP1 & 2 (Poly(RC) binding proteins 1 & 2; hnRNP E1 & E2), RBMX (RNA binding motif protein, X-linked; hnRNP G), PTB-1 (Polypyrimidine tract binding protein 1; hnRNP I), FUS (fused-in-sarcoma; hnRNP P2), SYNCRIP (Synaptotagmin binding cytoplasmic RNA interacting protein; hnRNP Q). However, a core subset of six hnRNPs (hnRNP A1, A2, B1, B2, C1, and C2) were found to be highly expressed, and consistently tightly associated with hnRNAs [9,20]. Of these, hnRNP A1 and A2 were most abundant, representing approximately 60% of the hnRNP mass in HeLa cells [9]. All six species were found to have affinity for nucleic acids, from single-stranded DNA (ssDNA) to poly-adenylated RNAs and purified hnRNAs. hnRNP C proteins demonstrated the highest affinity for RNAs, and an acidic pI of 5.9 [9]. In contrast, the hnRNP A and B proteins were distinguished by their highly basic pIs ranging from 8.2 to 9.6, with the additional unusual characteristics of high glycine content and enriched asymmetrical di-methylated arginine content [7,9,10]. Thus, without knowledge of their domain architecture, enzymatic activities, or function, the hnRNP A/B protein family (herein collectively referred to as A/B proteins) was defined. This review will focus upon the hnRNP A/B protein family members, specifically A1, A2/B1, A0, and A3, outlining their cellular function and highlighting their roles in disease pathogenesis. In particular, while the hnRNP A/B proteins share significant amino acid sequence identity (Figure1) and are all involved in multiple aspects of cellular RNA metabolism, there is clear evolutionary evidence (Figure2), supported by experimental data, that each protein fills a distinct role in cellular homeostasis. For example, all four proteins share a high N-terminal amino acid identity (Figure1C), but their C-terminal domains are more divergent in amino acid sequence and protein:protein interactors, facilitating their below-described distinct roles in the cell. To our knowledge, this is the first review to comprehensively address hnRNPs A0 and A3. It should be noted that: (1) a gene and protein product named HNRNPA1L2 (A1-like 2) that shares significant protein sequence similarity to the known hnRNP A/B proteins [21] is identified in NCBI, Uniprot, and Ensembl’s databases [22–24], but lacks any functional data, and (2) the confusingly-named HNRNPAB (protein: hnRNP AB or CBF-A) and CIRBP (protein: cold-inducible RNA binding protein, hnRNP A18) are evolutionarily distinct from the hnRNP A/B family proteins [25]; thus, these proteins will not be discussed further in this review. BiologyBiology2021 2021, 10, ,10 712, x FOR PEER REVIEW 3 of3 of41 40 FigureFigure 1. 1.Human Human hnRNP hnRNP A/B A/B proteins proteins areare closelyclosely relatedrelated and share similar alternative alternative exonic exonic structures. structures. Protein Protein sequences sequences fromfrom the the full-length full-length isoform isoform for for each each hnRNP hnRNP A/B A/B proteinprotein familyfamily membermember were aligned using using ClustalW ClustalW [26] [26].. (A (A) )Percent Percent identityidentity matrix matrix for for the the full-length full-length iso isoformsforms for for each each hnRNP hnRNP A/B A/B protein protein family family member member:: A0 (NCBI A0 (NCBI Protein Protein accession accession num- ber NP_006796.1); A1-B (NP_112420.1); B1 (NP_112533.1); A3-1 (NP_001317178.1). Boxes are shaded red to indicate degree number NP_006796.1); A1-B (NP_112420.1); B1 (NP_112533.1); A3-1 (NP_001317178.1). Boxes are shaded red to indicate of sequence identity. (B) Percent identity matrix for the most common isoforms for each hnRNP A/B protein family mem- degreeber, coloured of sequence as in identity.(A). A0 (NP_006796.1); (B) Percent identity A1-A (NP_002127.1);