RESUMEN
UV-crosslinking has proven to be an invaluable tool for the identification of RNA-protein interactomes. The paucity of methods for distinguishing background from bona fide RNA-protein interactions, however, makes attribution of RNA-binding function on UV-crosslinking alone challenging. To address this need, we previously reported an RNA-binding protein (RBP) confidence scoring metric (RCS), incorporating both signal-to-noise (S:N) and protein abundance determinations to distinguish high- and low-confidence candidate RBPs. Although RCS has utility, we sought a direct metric for quantification and comparative evaluation of protein-RNA interactions. Here we propose the use of protein-specific UV-crosslinking efficiency (%CL), representing the molar fraction of a protein that is crosslinked to RNA, for functional evaluation of candidate RBPs. Application to the HeLa RNA interactome yielded %CL values for 1097 proteins. Remarkably, %CL values span over five orders of magnitude. For the HeLa RNA interactome, %CL values comprise a range from high efficiency, high specificity interactions, e.g., the Elav protein HuR and the Pumilio homolog Pum2, with %CL values of 45.9 and 24.2, respectively, to very low efficiency and specificity interactions, for example, the metabolic enzymes glyceraldehyde-3-phosphate dehydrogenase, fructose-bisphosphate aldolase, and alpha-enolase, with %CL values of 0.0016, 0.006, and 0.008, respectively. We further extend the utility of %CL through prediction of protein domains and classes with known RNA-binding functions, thus establishing it as a useful metric for RNA interactome analysis. We anticipate that this approach will benefit efforts to establish functional RNA interactomes and support the development of more predictive computational approaches for RBP identification.
Asunto(s)
Proteínas de Unión al ARN , ARN , Rayos Ultravioleta , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/química , ARN/metabolismo , ARN/genética , Humanos , Células HeLa , Unión Proteica , Reactivos de Enlaces Cruzados/químicaRESUMEN
Signal recognition particle (SRP) pathway function in protein translocation across the endoplasmic reticulum (ER) is well established; its role in RNA localization to the ER remains, however, unclear. In current models, mRNAs undergo translation- and SRP-dependent trafficking to the ER, with ER localization mediated via interactions between SRP-bound translating ribosomes and the ER-resident SRP receptor (SR), a heterodimeric complex comprising SRA, the SRP-binding subunit, and SRB, an integral membrane ER protein. To study SRP pathway function in RNA localization, SR knockout (KO) mammalian cell lines were generated and the consequences of SR KO on steady-state and dynamic mRNA localization examined. CRISPR/Cas9-mediated SRPRB KO resulted in profound destabilization of SRA. Pairing siRNA silencing of SRPRA in SRPRB KO cells yielded viable SR KO cells. Steady-state mRNA compositions and ER-localization patterns in parental and SR KO cells were determined by cell fractionation and deep sequencing. Notably, steady-state cytosol and ER mRNA compositions and partitioning patterns were largely unaltered by loss of SR expression. To examine SRP pathway function in RNA localization dynamics, the subcellular trafficking itineraries of newly exported mRNAs were determined by 4-thiouridine (4SU) pulse-labeling/4SU-seq/cell fractionation. Newly exported mRNAs were distinguished by high ER enrichment, with ER localization being SR-independent. Intriguingly, under conditions of translation initiation inhibition, the ER was the default localization site for all newly exported mRNAs. These data demonstrate that mRNA localization to the ER can be uncoupled from the SRP pathway function and reopen questions regarding the mechanism of RNA localization to the ER.
Asunto(s)
Retículo Endoplásmico , Partícula de Reconocimiento de Señal , Animales , ARN Mensajero/genética , ARN Mensajero/metabolismo , Partícula de Reconocimiento de Señal/genética , Partícula de Reconocimiento de Señal/metabolismo , Retículo Endoplásmico/genética , Retículo Endoplásmico/metabolismo , Ribosomas/genética , Ribosomas/metabolismo , Citosol/metabolismo , Proteínas de la Membrana/genética , Mamíferos/genéticaRESUMEN
Synonymous mutations do not alter the specified amino acid but may alter the structure or function of an mRNA in ways that impact fitness. There are few examples in the literature, however, in which the effects of synonymous mutations on microbial growth rates have been measured, and even fewer for which the underlying mechanism is understood. We evolved four populations of a strain of Salmonella enterica in which a promiscuous enzyme has been recruited to replace an essential enzyme. A previously identified point mutation increases the enzyme's ability to catalyze the newly needed reaction (required for arginine biosynthesis) but decreases its ability to catalyze its native reaction (required for proline biosynthesis). The poor performance of this enzyme limits growth rate on glucose. After 260 generations, we identified two synonymous mutations in the first six codons of the gene encoding the weak-link enzyme that increase growth rate by 41 and 67%. We introduced all possible synonymous mutations into the first six codons and found substantial effects on growth rate; one doubles growth rate, and another completely abolishes growth. Computational analyses suggest that these mutations affect either the stability of a stem-loop structure that sequesters the start codon or the accessibility of the region between the Shine-Dalgarno sequence and the start codon. Thus, these mutations would be predicted to affect translational efficiency and thereby indirectly affect mRNA stability because translating ribosomes protect mRNA from degradation. Experimental data support these hypotheses. We conclude that the effects of the synonymous mutations are due to a combination of effects on mRNA stability and translation efficiency that alter levels of the weak-link enzyme. These findings suggest that synonymous mutations can have profound effects on fitness under strong selection and that their importance in evolution may be under-appreciated.
Asunto(s)
Proteínas Bacterianas/genética , Aptitud Genética , ARN Mensajero/genética , Salmonella enterica/crecimiento & desarrollo , Mutación Silenciosa , Codón , Variaciones en el Número de Copia de ADN , Evolución Molecular , Conformación de Ácido Nucleico , Operón , Fosfotransferasas (aceptor de Grupo Carboxilo)/genética , Fosfotransferasas (aceptor de Grupo Carboxilo)/metabolismo , Mutación Puntual , Proteómica , Estabilidad del ARN , Ribosomas/genética , Salmonella enterica/genética , Secuenciación Completa del GenomaRESUMEN
The critical roles of RNA-binding proteins (RBPs) in all aspects of RNA biology fostered the development of methods utilizing ultraviolet (UV) crosslinking and method-specific RNA enrichment steps for proteome-wide identification and assessment of RBP function. Despite the substantial contributions of these UV-based RNA-centric methods to our understanding of RNA-protein interaction networks, their utility is constrained by biases in RBP recovery and significant noise contributions, which can confound meaningful interpretation. To overcome these issues, we recently developed a method termed Liquid Emulsion-Assisted Purification of RNA-Bound Protein (LEAP-RBP) and introduced quantitative signal-to-noise (S:N)-based metrics for the proteome-wide identification of RNA interactomes and accurate assessment of global RBP occupancy dynamics. Compared to existing methodologies, LEAP-RBP provides significant advantages in speed, cost, efficiency, and selectivity for RNA-bound proteins. In this work, we provide a step-by-step guide for the successful application of the LEAP-RBP method for both small- and large-scale investigations of RNA-bound proteomes. Key features ⢠Unbiased and efficient isolation of total RNA-bound protein, RNA, and protein from biological samples. ⢠Cost-effective identification of proteome-wide RNA interactomes and validation of direct RNA-binding protein functionality. ⢠Robust and accurate assessment of context- and/or condition-dependent RBP occupancy state dynamics.
RESUMEN
Recent efforts towards the comprehensive identification of RNA-bound proteomes have revealed a large, surprisingly diverse family of candidate RNA-binding proteins (RBPs). Quantitative metrics for characterization and validation of protein-RNA interactions and their dynamic interactions have, however, proven analytically challenging and prone to error. Here we report a method termed LEAP-RBP (Liquid-Emulsion-Assisted-Purification of RNA-Bound Protein) for the selective, quantitative recovery of UV-crosslinked RNA-protein complexes. By virtue of its high specificity and yield, LEAP-RBP distinguishes RNA-bound and RNA-free protein levels and reveals common sources of experimental noise in RNA-centric RBP enrichment methods. We introduce strategies for accurate RBP identification and signal-based metrics for quantifying protein-RNA complex enrichment, relative RNA occupancy, and method specificity. In this work, the utility of our approach is validated by comprehensive identification of RBPs whose association with mRNA is modulated in response to global mRNA translation state changes and through in-depth benchmark comparisons with current methodologies.