Interactions of all sorts make up much of biology. Ongoing research, for example, reveals that RNA and proteins work together in more ways than most biologists might have expected. “It is now well established that post-transcriptional regulation—encompassing a diversity of non-coding RNAs and RNA-binding proteins (RBPs) as major players—is at least as important as transcriptional regulation for a multitude of biological processes,” explains Bruno Pereira, post-doctoral researcher in the differentiation and cancer group at the University of Porto.
“An interesting research area is related to the impact RBPs might have in the rewiring of cellular metabolic programs, as it is becoming evident that a number of metabolic enzymes also possess RNA-binding activity. These dual function proteins might be very relevant in the direct regulation of distinctive metabolic profiles,” Pereira adds.
The amount of genome related to RBPs also speaks to the expanse of the implications of these molecules. “Roughly 8% of the protein-coding genes in humans encode polypeptides that directly bind or process RNA,” says Auinash Kalsotra, an assistant professor in the department of biochemistry at the University of Illinois. These RBPs control the metabolism of RNA from start to finish. “It was known for a long time that—in addition to the canonical RNA-binding domains that form stable secondary and tertiary structures—many RBPs also contain intrinsically disordered low amino acid complexity (LC) domains that lack a well-defined three-dimensional structure,” Kalsotra says. “Yet, the exact roles of these LC domains within RBPs were not well understood.”
That is starting to change. As Kalsotra points out, recent research shows that LC domains in RBPs “enable transient multivalent interactions between soluble RNA and protein molecules promoting liquid-liquid phase transitions that underlie biogenesis of various membrane-less organelles.” Beyond the interests that these discoveries have generated in cellular and molecular biology, mutations in RBPs appear to be involved in neurodegenerative disorders. “Mutations in LC domains predispose RBPs to misfold and aggregate, which is now considered as the common pathogenic feature of neurodegenerative diseases, including amyotrophic lateral sclerosis, frontotemporal dementia, as well as multisystem proteinopathy,” Kalsotra explains.
In all likelihood, what scientists have to learn about the importance of RNA-protein interactions in general is just getting started.
Exploring the interactions
To learn more about RNA-protein interactions, scientists need tools. “Some of the most interesting advances are related to the introduction of novel techniques to identify RBP targets that don’t rely on immunoprecipitation (IP) strategies, which have been the classical approach so far,” Pereira says. “These broaden up the existing tool box, as in several cases you might be hampered by the lack of sufficiently good quality antibodies to perform RNA-IP methods.”
“These new methodologies are built from fusion proteins composed of an RBP of interest and catalytic domains of specific RNA-editing enzymes that introduce permanent marks in the RNA targets that can then be identified by simply sequencing them,” Pereira adds.
Exploring more broadly will teach us even more about the impact of RBPs on RNA processing.
Exploring more broadly will teach us even more about the impact of RBPs on RNA processing. “Combinations of genome-wide approaches have provided significant advances in studying how and which RBPs regulate particular RNA processing events in individual cells or tissues,” Kalsotra says. “These include whole-transcriptome analysis to measure ratios of processed versus unprocessed RNAs after the loss- or gain-of-function studies—through RNA interference, CRISPR/genetic knockout or overexpression—along with genome-wide identification of binding sites of particular RBPs in the target cell type.”
The way that RBPs impact processes also matters. “Hundreds of RNA processing events are typically sensitive to the overall concentrations of an RBP in a cell,” Kalsotra says. “Some of these events are regulated by the RBP through direct protein-RNA interactions, and others are due to secondary effects.” Scientists want to explore these events in vitro and in vivo in global ways. To do that, Kalsotra points out the value of techniques like cross-linking immunoprecipitation (CLIP), which is an antibody-based method that uses UV radiation to cross-link an RBP with its RNA target.
Following CLIP with next-generation sequencing, RNAcompete, RNA bind-n-seq, RNA array, and HiTS-RAP, says Kalsotra, paves “innovative ways to globally measure functional RBP binding to RNA, determine the most preferred sequence motif or motifs for a given RBP, measure the kinetics of RBP–RNA interactions under specific settings– and compare the importance of RNA secondary structure to the sequence context for RBP-RNA interactions.”
Expanding the knowledge
When asked about the most interesting recent discovery about RBPs, William Lee, vice president of operations at EpiGentek, says it’s that they provide “rapid and dynamic post-transcriptional regulation for control of gene expression.”
The more that biologists know about how RBPs work, the more scientists can learn from these processes, and, possibly, how to use them in healthcare. “Most RBPs recognize short—5 to 8 nucleotide long—degenerate RNA motifs,” Kalsotra says. “Therefore, RNA sequence alone is not always sufficient to guide protein binding, which means additional features must specify RBP-RNA interactions within cells.”
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Scientists can explore the principles behind RBP-RNA interactions more closely than ever, Kalsotra notes, through two exciting advances. “First, the epitranscriptome seems to greatly expand the information encoded by RNA,” Kalsotra says. “These RNA modifications are dynamic, reversible, and dictate protein-RNA interactions in response to various physiological stimuli.”
The second advance that Kalsotra points out is the development of new transcriptome-wide approaches—DMS-seq, icSHAPE, PARIS, hiCLIP—to study RNA structures in vivo have uncovered the importance of RNA structures in shaping the RBP binding to RNAs. As an example, he says, “Structural rearrangements during different phases of the RNA life cycle seem to facilitate remodeling of the RNA-protein complexes through the occlusion, exposure, or formation of RBP-binding sites.”
The breadth of RBP-related impacts remains to be entirely seen. As Lee points out, scientists will study “genome-wide interactions of RBPs by next-generation sequencing combined with computational methods to analyze experimental data and to obtain insights into the specificity of protein-RNA interactions.” Those insights will influence how biologists think about molecular mechanisms in the future.
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