RBP Human, Native

Basic Research Questions

• What are RNA-binding proteins (RBPs) and why are they important in human biology?

RNA-binding proteins are cellular components that interact with RNA molecules through specialized domains. Mammalian cells harbor more than a thousand RNA-binding proteins, with half of these employing unknown modes of RNA binding . These proteins regulate every aspect of RNA metabolism and function, from transcription to translation and degradation.

The importance of RBPs in human biology is highlighted by several factors:

• They control gene expression at the post-transcriptional level

• They organize ribonucleoprotein complexes essential for cellular function

• Their dysregulation is implicated in numerous human diseases

• They provide additional regulatory layers beyond transcriptional control

Methodologically, studying RBPs requires approaches that can preserve native interactions while providing molecular-level resolution of binding regions and dynamics.

• How many RNA-binding proteins have been identified in human cells and how are they classified?

Current research indicates that mammalian cells contain more than a thousand RNA-binding proteins . These proteins can be classified based on several parameters:

Comprehensive studies using RBDmap have identified 1,174 binding sites within 529 HeLa cell RBPs, significantly expanding our understanding of the RNA-binding proteome .

• What RNA-binding domains (RBDs) have been identified in human proteins and how are they characterized?

RNA-binding domains are protein regions that directly contact RNA molecules. Recent methodological advances have expanded our understanding beyond classical RBDs:

• UV-crosslinking proteins to RNA in living cells

• Capturing polyadenylated RNAs with oligo(dT)

• Partial proteolysis to generate RNA-bound peptides

• Second capture step to isolate RNA-bound fragments

• Mass spectrometry identification of these fragments

This methodological approach revealed that many RBDs coincide with:

• Catalytic centers of enzymes

• Protein-protein interaction domains

• Intrinsically disordered regions

RBDmap validation showed that 70.3% (with LysC digestion) and 81% (with ArgC digestion) of identified RNA-binding peptides are proximal to RNA in known protein-RNA co-structures, demonstrating the high specificity of this approach .

• How do canonical and non-canonical RBPs differ in their RNA-binding mechanisms?

The distinction between canonical and non-canonical RBPs lies in their RNA-recognition mechanisms and evolutionary history:

Many newly discovered RNA-binding proteins do not show architectural similarities with classical RBPs, and their modes of interaction with RNA remained unclear until the development of methods like RBDmap . Research has now revealed that non-canonical RBPs often bind RNA through:

• Enzymatic active sites, suggesting RNA may regulate their catalytic activities

• Protein-protein interaction interfaces, indicating RNA could modulate protein complex formation

• Intrinsically disordered regions that provide conformational flexibility

This methodological insight into non-canonical RBPs reveals additional regulatory layers where RNA binding could modulate the primary functions of these proteins .

Advanced Research Questions

• What experimental methods are available for identifying RBP-RNA interactions in native human cells?

Several complementary methods have been developed to capture RBP-RNA interactions in their native context:

RAPseq enables in vitro large-scale profiling of RBP binding to native RNAs and has been used to study the evolution of HUR across vertebrates, revealing that it binds predominantly to introns in zebrafish compared to 3'UTRs in human RNAs . Co-RAPseq uncovered cooperative RNA binding of HUR and PTBP1 within an optimal distance of 27 nucleotides .

RBDmap identified 1,174 high-confidence RNA-binding sites within 529 proteins by combining UV crosslinking, oligo(dT) capture, proteolytic digestion, and a second oligo(dT) capture .

ARTR-seq avoids ultraviolet crosslinking and immunoprecipitation, allowing for efficient and specific identification of RBP binding sites from as few as 20 cells or a tissue section .

• How do intrinsically disordered regions contribute to RNA binding in human RBPs?

Intrinsically disordered regions (IDRs) have emerged as prevalent partners in protein-RNA interactions:

Nearly half of the RNA-binding sites identified by RBDmap map to intrinsically disordered regions, uncovering unstructured domains as prevalent partners in protein-RNA interactions . For 170 RBPs, a disordered RBD was identified as the sole detectable RNA-binding site .

Methodologically, these findings required approaches like RBDmap that do not depend on structural information or sequence conservation, as disordered regions are often poorly conserved at the sequence level despite functional conservation.

Disordered RBDpeps largely mirror the chemical properties of the whole RBDpep superset, apart from the expected enrichment for disorder-promoting residues (proline, serine, and glycine), as well as arginine and glutamine . The flexibility of these regions may allow for adaptable binding to different RNA targets and provide opportunities for regulation through post-translational modifications.

• What is the role of post-translational modifications in regulating RBP function?

Post-translational modifications (PTMs) provide a dynamic regulatory layer for RBP function:

Research has shown that RNA-binding sites represent hot spots for defined posttranslational modifications such as lysine acetylation and tyrosine phosphorylation, suggesting metabolic and signal-dependent regulation of RBP function .

Methodologically, studying PTMs in RBPs requires:

• Identification of modification sites (mass spectrometry)

• Determination of their prevalence (quantitative proteomics)

• Functional characterization (mutagenesis, binding assays)

• Identification of enzymes responsible (kinases, acetylases, etc.)

The enrichment of PTM sites within RNA-binding regions suggests evolutionary selection for regulation through modifications. This creates a dynamic regulatory network where cellular signaling can rapidly modulate RNA metabolism through RBP modifications without altering protein levels.

• How can researchers study dynamic changes in RBP-RNA interactions?

Capturing the dynamic nature of RBP-RNA interactions requires specialized methodological approaches:

ARTR-seq takes advantage of rapid formaldehyde fixation to capture dynamic RNA binding by RBPs over a short period of time, enabling temporal studies of RBP-RNA interactions . This method avoids ultraviolet crosslinking and immunoprecipitation, which can limit the types of interactions that are captured.

For studying dynamics methodologically:

• Establish appropriate time points based on the process of interest

• Apply fixation methods that rapidly preserve interactions (formaldehyde for ARTR-seq)

• Use consistent extraction and processing protocols across time points

• Apply statistical methods designed for time-series data

• Validate dynamic changes using orthogonal approaches

This approach is particularly valuable for understanding how RBP-RNA interactions change during cellular processes like differentiation, stress response, or signaling pathway activation.

• What computational approaches are most effective for predicting RBP binding sites?

Computational prediction of RBP binding sites combines multiple data types and algorithms:

Effective computational approaches typically integrate:

• Primary sequence preferences derived from experimental data

• RNA structural features (from SHAPE-seq or similar)

• Evolutionary conservation information

• Data from high-throughput binding assays like RBDmap

Methodologically, researchers should:

• Train models on high-quality, diverse datasets

• Incorporate both positive and negative examples

• Validate predictions with orthogonal experimental approaches

• Consider the biological context (cell type, conditions) of the original training data

As methods like RAPseq , RBDmap , and ARTR-seq generate more comprehensive datasets, computational predictions are becoming increasingly accurate and biologically relevant.

• How do disease-associated mutations in RBPs impact their function and contribute to pathology?

Disease-associated mutations in RBPs can disrupt RNA regulation through various mechanisms:

Research on pathological IGF2BP family variants showed that five disease-associated mutations exhibited different RNA binding patterns compared to wild-type protein . This demonstrates how mutations can directly affect the RBP-RNA interactome.

RBDs display a high degree of evolutionary conservation and incidence of Mendelian mutations, suggestive of important functional roles . This evolutionary constraint indicates that mutations in these regions are likely to have significant functional consequences.

Methodologically, studying disease-associated mutations requires:

• Identification of mutations (patient sequencing)

• Functional characterization (RAPseq or similar)

• Cellular phenotyping (RNA-seq to assess target regulation)

• Animal or organoid models to understand tissue-specific effects

Mutations in RBPs that disrupt RNA binding can lead to widespread dysregulation of RNA processing, contributing to disease through both loss-of-function and gain-of-function mechanisms.