Abstract:Eukaryotic cells express a large variety of RNA binding proteins (RBPs), with diverse affinities and specificities towards target RNAs. These proteins play a crucial role in almost every aspect of RNA biogenesis, expression and function. The heterogeneous nuclear ribonucleoproteins (hnRNPs) are a complex and diverse family of RNA binding proteins. hnRNPs display multiple functions in the processing of heterogeneous nuclear RNAs into mature messenger RNAs. hnRNP A1 is one of the most abundant and ubiquitously e… Show more
“…The prototypical member of this family, hnRNP A1, regulates alternative splicing, nuclear export, translation, and other RNA processing events (17). HnRNP A1 has a modular domain organization consisting of tandem RNA recognition motifs (RRMs), collectively referred to as unwinding protein 1 (UP1), and an intrinsically disordered RGG-rich C terminus.…”
mentioning
confidence: 99%
“…Methods such as high-throughput sequencing kinetics (HTS-KIN), RNA Bind-n-Seq, and RNA MaP have recently been developed that allow the affinities and reaction kinetics of thousands of RNAs to be measured simultaneously (6,(12)(13)(14)(15). These methods can potentially provide global information on the surrounding context of a given sequence motif; however, they have yet to see wide application for structure/function studies of RNA specificity.Heterogeneous nuclear ribonucleoproteins (hnRNPs) represent a diverse family of RBPs that are implicated at most stages of posttranscriptional gene regulation (16,17). The prototypical member of this family, hnRNP A1, regulates alternative splicing, nuclear export, translation, and other RNA processing events (17).…”
mentioning
confidence: 99%
“…Heterogeneous nuclear ribonucleoproteins (hnRNPs) represent a diverse family of RBPs that are implicated at most stages of posttranscriptional gene regulation (16,17). The prototypical member of this family, hnRNP A1, regulates alternative splicing, nuclear export, translation, and other RNA processing events (17).…”
Heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) is a multipurpose RNA-binding protein (RBP) involved in normal and pathological RNA metabolism. Transcriptome-wide mapping and in vitro evolution identify consensus hnRNP A1 binding motifs; however, such data do not reveal how surrounding RNA sequence and structural context modulate affinity. We determined the affinity of hnRNP A1 for all possible sequence variants (n = 16,384) of the HIV exon splicing silencer 3 (ESS3) 7-nt apical loop. Analysis of the affinity distribution identifies the optimal motif 5′-YAG-3′ and shows how its copy number, position in the loop, and loop structure modulate affinity. For a subset of ESS3 variants, we show that specificity is determined by association rate constants and that variants lacking the minimal sequence motif bind competitively with consensus RNA. Thus, the results reveal general rules of specificity of hnRNP A1 and provide a quantitative framework for understanding how it discriminates between alternative competing RNA ligands in vivo.hnRNP A1 | protein-RNA specificity | RNA structure | thermodynamics | binding kinetics G ene expression is regulated by an ensemble of protein-RNA complexes that assemble and disassemble throughout the lifetime of a transcript (1-5). Knowledge of the determinants of RNA-binding protein (RBP) specificity is therefore essential to understanding the relative affinity for sites of association within the transcriptome. A characteristic feature of many RBPs is their ability to elicit biological function by binding at sites in RNAs that vary significantly in sequence and local structure. This broad specificity of RBPs challenges the simplistic description of RNA binding as either specific or nonspecific. Global profiling methods provide consensus sequence motifs that represent preferential binding sites by RBPs in the transcriptome (6-11). Although powerful, such approaches are usually nonquantitative. Moreover, they do not readily provide information on how surrounding sequence identity or positioning of preferred sequences within higher order structure alters affinity. Methods such as high-throughput sequencing kinetics (HTS-KIN), RNA Bind-n-Seq, and RNA MaP have recently been developed that allow the affinities and reaction kinetics of thousands of RNAs to be measured simultaneously (6,(12)(13)(14)(15). These methods can potentially provide global information on the surrounding context of a given sequence motif; however, they have yet to see wide application for structure/function studies of RNA specificity.Heterogeneous nuclear ribonucleoproteins (hnRNPs) represent a diverse family of RBPs that are implicated at most stages of posttranscriptional gene regulation (16,17). The prototypical member of this family, hnRNP A1, regulates alternative splicing, nuclear export, translation, and other RNA processing events (17). HnRNP A1 has a modular domain organization consisting of tandem RNA recognition motifs (RRMs), collectively referred to as unwinding protein 1 (UP1), and an intrinsically di...
“…The prototypical member of this family, hnRNP A1, regulates alternative splicing, nuclear export, translation, and other RNA processing events (17). HnRNP A1 has a modular domain organization consisting of tandem RNA recognition motifs (RRMs), collectively referred to as unwinding protein 1 (UP1), and an intrinsically disordered RGG-rich C terminus.…”
mentioning
confidence: 99%
“…Methods such as high-throughput sequencing kinetics (HTS-KIN), RNA Bind-n-Seq, and RNA MaP have recently been developed that allow the affinities and reaction kinetics of thousands of RNAs to be measured simultaneously (6,(12)(13)(14)(15). These methods can potentially provide global information on the surrounding context of a given sequence motif; however, they have yet to see wide application for structure/function studies of RNA specificity.Heterogeneous nuclear ribonucleoproteins (hnRNPs) represent a diverse family of RBPs that are implicated at most stages of posttranscriptional gene regulation (16,17). The prototypical member of this family, hnRNP A1, regulates alternative splicing, nuclear export, translation, and other RNA processing events (17).…”
mentioning
confidence: 99%
“…Heterogeneous nuclear ribonucleoproteins (hnRNPs) represent a diverse family of RBPs that are implicated at most stages of posttranscriptional gene regulation (16,17). The prototypical member of this family, hnRNP A1, regulates alternative splicing, nuclear export, translation, and other RNA processing events (17).…”
Heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) is a multipurpose RNA-binding protein (RBP) involved in normal and pathological RNA metabolism. Transcriptome-wide mapping and in vitro evolution identify consensus hnRNP A1 binding motifs; however, such data do not reveal how surrounding RNA sequence and structural context modulate affinity. We determined the affinity of hnRNP A1 for all possible sequence variants (n = 16,384) of the HIV exon splicing silencer 3 (ESS3) 7-nt apical loop. Analysis of the affinity distribution identifies the optimal motif 5′-YAG-3′ and shows how its copy number, position in the loop, and loop structure modulate affinity. For a subset of ESS3 variants, we show that specificity is determined by association rate constants and that variants lacking the minimal sequence motif bind competitively with consensus RNA. Thus, the results reveal general rules of specificity of hnRNP A1 and provide a quantitative framework for understanding how it discriminates between alternative competing RNA ligands in vivo.hnRNP A1 | protein-RNA specificity | RNA structure | thermodynamics | binding kinetics G ene expression is regulated by an ensemble of protein-RNA complexes that assemble and disassemble throughout the lifetime of a transcript (1-5). Knowledge of the determinants of RNA-binding protein (RBP) specificity is therefore essential to understanding the relative affinity for sites of association within the transcriptome. A characteristic feature of many RBPs is their ability to elicit biological function by binding at sites in RNAs that vary significantly in sequence and local structure. This broad specificity of RBPs challenges the simplistic description of RNA binding as either specific or nonspecific. Global profiling methods provide consensus sequence motifs that represent preferential binding sites by RBPs in the transcriptome (6-11). Although powerful, such approaches are usually nonquantitative. Moreover, they do not readily provide information on how surrounding sequence identity or positioning of preferred sequences within higher order structure alters affinity. Methods such as high-throughput sequencing kinetics (HTS-KIN), RNA Bind-n-Seq, and RNA MaP have recently been developed that allow the affinities and reaction kinetics of thousands of RNAs to be measured simultaneously (6,(12)(13)(14)(15). These methods can potentially provide global information on the surrounding context of a given sequence motif; however, they have yet to see wide application for structure/function studies of RNA specificity.Heterogeneous nuclear ribonucleoproteins (hnRNPs) represent a diverse family of RBPs that are implicated at most stages of posttranscriptional gene regulation (16,17). The prototypical member of this family, hnRNP A1, regulates alternative splicing, nuclear export, translation, and other RNA processing events (17). HnRNP A1 has a modular domain organization consisting of tandem RNA recognition motifs (RRMs), collectively referred to as unwinding protein 1 (UP1), and an intrinsically di...
“…In fact, the largest grouping of proteins unique to A1-EVs (Figure 7(b)) is involved in protein metabolism (31%), and includes extracellular matrix remodelling [22,48] as well as proteosomal and ribosomal subunit factors (Figure 7(c,d)). The second highest proportion of proteins unique to A1-EVs are nucleic acid metabolism factors, with particular enrichment of heterogeneous nuclear ribonucleoprotein family members, responsible for RNA shuttling (Figure 7(d)) [49,50]. Similar protein profiles were seen using mass spectrometry and Western blot with A1-EVs enriched by either UFC or precipitation methods (Supplemental Figure 3 and Supplemental Tables 1 and 2).10.1080/20013078.2018.1456888-F0007Figure 7.EV protein profiles as determined by mass spectrometry.…”
Newts can regenerate amputated limbs and cardiac tissue, unlike mammals which lack broad regenerative capacity. Several signaling pathways involved in cell proliferation, differentiation and survival during newt tissue regeneration have been elucidated, however the factors that coordinate signaling between cells, as well as the conservation of these factors in other animals, are not well defined. Here we report that media conditioned by newt limb explant cells (A1 cells) protect mammalian cardiomyocytes from oxidative stress-induced apoptosis. The cytoprotective effect of A1-conditioned media was negated by exposing A1 cells to GW4869, which suppresses the generation of extracellular vesicles (EVs). A1-EVs are similar in diameter (~100–150 nm), structure, and share several membrane surface and cargo proteins with mammalian exosomes. However, isolated A1-EVs contain significantly higher levels of both RNA and protein per particle than mammalian EVs. Additionally, numerous cargo RNAs and proteins are unique to A1-EVs. Of particular note, A1-EVs contain numerous mRNAs encoding nuclear receptors, membrane ligands, as well as transcription factors. Mammalian cardiomyocytes treated with A1-EVs showed increased expression of genes in the PI3K/AKT pathway, a pivotal player in survival signaling. We conclude that newt cells secrete EVs with diverse, distinctive RNA and protein contents. Despite ~300 million years of evolutionary divergence between newts and mammals, newt EVs confer cytoprotective effects on mammalian cardiomyocytes.
“…The hnRNP proteins share common structural domains and have central roles in regulating gene expression at both transcriptional and translational levels [11]. They also are involved in RNA splicing, telomere biogenesis, DNA repair and cell signaling [12]. Several recent reports show a role for individual hnRNPs in tumor development and progression in a variety of cancers [13][14][15].…”
Clinical and experimental data suggest that carcinoembryonic antigen (CEA, CD66e, CEACAM-5) plays a key role in the formation of hepatic metastasis from colorectal and other types of epithelial cancers. The molecular events involved in CEA-induced metastasis have yet to be defined. Our group first cloned the gene (CEAR) for CEAbinding protein from the surface of fixed liver macrophages, (Kupffer cells). In this study to further elucidate the role of CEAR in colorectal cancer progression, its expression in colorectal cancer cells was suppressed by short hairpin RNAs (shRNAs) in CEA-overexpressing and CEA -negative MIP-101 colorectal cancer cell lines. The data show that targeted suppression of endogenous CEAR in tumor cells resulted in changes in cell invasiveness. RT-PCR data indicated reduced levels of E-cadherin, Snail, MMP-2, and Oct-4 in the clones with suppressed CEAR suggesting a role in the epithelial mesenchymal transition. The comparative analysis of tumorigenic activity to the liver of the cell lines with suppressed CEAR has also been conducted using an intrasplenic injection model in immuno-deficient mice. This data shows a decrease in tumor progression associated with CEAR suppression. In summary the results of this study revealed a novel role for CEAR gene in the regulation of colorectal cancer cell invasiveness and progression.
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