Molecular Genetic Analysis of Rbm45/Drbp1: Genomic Structure, Expression, and Evolution

Abstract

RNA recognition motif-type RNA-binding domain containing proteins (RBDPs) participate in RNA metabolism including regulating mRNA stability, nuclear-cytoplasmic shuttling, and splicing. Rbm45 is an RBDP first cloned from rat brain and expressed spatiotemporally during rat neural development. More recently, RBM45 has been associated with pathological aggregates in the human neurological disorders amyotrophic lateral sclerosis, frontotemporal lobar degeneration, and Alzheimer’s. Rbm45 and the neural developmental protein musashi-1 are in the same family of RDBPs and have similar expression patterns. In contrast to Musashi-1, which is upregulated during colorectal carcinogenesis, we found no association of RBM45 overexpression in human colon cancer tissue. In order to begin characterizing RNA-binding partners of Rbm45, we have successfully cloned and expressed human RBM45 in an Intein fusion-protein expression system. Furthermore, to gain a better understanding of the molecular genetics and evolution of Rbm45, we used an in silico approach to analyze the gene structure of the human and mouse Rbm45 homologues and explored the evolutionary conservation of Rbm45 in metazoans. Human RBM45 and mouse Rbm45 span ~17 kb and 13 kb, respectively, and contain 10 exons, one of which is non-coding. Both genes have TATA-less promoters with an initiator and a GC-rich element. Downstream of exon 10, both homologues have canonical polyadenylation signals and an embryonic cytoplasmic polyadenylation element. Moreover, our data indicate Rbm45 is conserved across all metazoan taxa from sponges (phylum Porifera) to humans (phylum Chordata), portending a fundamental role in metazoan development.

Rbm45 Phylogenetics, Protein Domain Conservation, and Gene Architecture in Clade Metazoa

“…Our lab [26] has previously demonstrated that nucleotide and amino acid phylograms of 10 vertebrate Rbm45 orthologues recapitulate accepted taxonomic relationships between classes Actinopterygii (ray-finned fishes), Amphibia, Reptilia, and Mammalia; additionally, through NCBI database interrogation, we reported Rbm45 orthologues, both empirically confirmed and Gnomon algorithm predicted (https://www.ncbi.nlm.nih.gov/genome/annotation_euk/process/), across metazoan taxa including animals from the non-Bilateria phyla Porifera (sponges) and Cnidaria (e.g., hydra), and within clade Bilateria phyla from the nephrozoan lineages Protostomia (e.g., phyla Mollusca and Arthropoda) and Deuterostomia (e.g., phyla Echinodermata and Chordata). Molecular phylogenetic analysis has been successfully used to reconstruct the evolutionary history of populations, genes, and proteins; furthermore, it has been utilized to understand genome organization and gene conservation [81,82].…”

“…NCBI gene annotation (https://www.ncbi.nlm.nih.gov/homologene/?term=Rbm45 [accessed 2024 February 4]), our previous work [26], and data from Tamada et al [27] reveal that Rbm45 contains four RBDs. However, ensuing work by two leading Rbm45 research teams show an alternative structure with three RBDs [31,32]; for consistency, we have adopted these research team’s Rbm45 domain nomenclature for this study.…”

“…These data implicate Rbm45 in neurogenesis, neural repair, and neuropathophysiology. We [26] and others [31,32] have shown that Rbm45 is highly conserved across metazoan taxa; intriguingly, our previous work [26] identified an Rbm45 orthologue in sponges (phylum Porifera) which lack a nervous system.…”

Rbm45 Phylogenetics, Protein Domain Conservation, and Gene Architecture in Clade Metazoa

“…Rbm45 is an ancient gene, conserved from sponges to humans [26]. The human RBM45 locus on chromosome 2q31.2 [26] encodes a promiscuous RBDP found to be expressed under normal physiological conditions predominantly in neuronal tissue of rats [27], mice [26,28], and humans [29,30].…”

“…Rbm45 is an ancient gene, conserved from sponges to humans [26]. The human RBM45 locus on chromosome 2q31.2 [26] encodes a promiscuous RBDP found to be expressed under normal physiological conditions predominantly in neuronal tissue of rats [27], mice [26,28], and humans [29,30]. Rbm45 has three canonical RNA-binding domains (RBD I, II, III) [27,31,32] that preferentially bind GC-rich RNA sequence motifs [27,33,34] and have recently been shown to facilitate binding to single-stranded DNA [35]; additionally, N-terminal RBD I and RBD II have been demonstrated to participate in RNA-independent protein-protein interactions [36].…”