High-throughput mapping of modular regulatory domains in human RNA-binding proteins.

RNA regulation in immunity.

Transcriptome-wide Analysis of Regulatory Interactions of the RNA-Binding Protein HuR

RNA binding proteins (RBPs) play essential roles in the regulation of RNA metabolism. Recent studies have disclosed that RBPs achieve their functions via binding to their targets in a position-dependent pattern on RNAs. However, few studies have systematically addressed the associations between the RBP's functions and their positional binding preferences. Here, we present large-scale analyses on the functional targets of human RBPs by integrating the enhanced cross-linking and immunoprecipitation followed by sequencing (eCLIP-seq) datasets and the shRNA knockdown followed by RNA-seq datasets that are deposited in the integrated ENCyclopedia of DNA Elements in the human genome (ENCODE) data portal. We found that (1) binding to the translation termination site and the 3'untranslated region is important to most human RBPs in the RNA decay regulation; (2) RBPs' binding and regulation follow a cell-type specific pattern. These analysis results show the strong relationship between the binding position and the functions of RBPs, which provides novel insights into the RBPs' regulation mechanisms.

Principles and Properties of Eukaryotic mRNPs

Modular architecture and functional annotation of human RNA-binding proteins containing RNA recognition motif

HuR and TTP: Two RNA Binding Proteins That Deliver Message From the 3′ End

Argonaute is the primary mediator of metazoan miRNA targeting (MT). Among the currently identified >1,500 human RNA-binding proteins (RBPs), there are only a handful of RBPs known to enhance MT and several others reported to suppress MT, leaving the global impact of RBPs on MT elusive. In this study, we have systematically analyzed transcriptome-wide binding sites for 150 human RBPs and evaluated the quantitative effect of individual RBPs on MT efficacy. In contrast to previous studies, we show that most RBPs significantly affect MT and that all of those MT-regulating RBPs function as MT enhancers rather than suppressors, by making the local secondary structure of the target site accessible to Argonaute. Our findings illuminate the unappreciated regulatory impact of human RBPs on MT, and as these RBPs may play key roles in the gene regulatory network governed by metazoan miRNAs, MT should be understood in the context of co-regulating RBPs.

Identifying binding targets of RNA-binding proteins (RBPs) can greatly facilitate our understanding of their functional mechanisms. Most computational methods employ machine learning to train classifiers on either RBP-specific targets or pooled RBP–RNA interactions. The former strategy is more powerful, but it only applies to a few RBPs with a large number of known targets; conversely, the latter strategy sacrifices prediction accuracy for a wider application, since specific interaction features are inevitably obscured through pooling heterogeneous datasets. Here, we present beRBP, a dual approach to predict human RBP–RNA interaction given PWM of a RBP and one RNA sequence. Based on Random Forests, beRBP not only builds a specific model for each RBP with a decent number of known targets, but also develops a general model for RBPs with limited or null known targets. The specific and general models both compared well with existing methods on three benchmark datasets. Notably, the general model achieved a better performance than existing methods on most novel RBPs. Overall, as a composite solution overarching the RBP-specific and RBP-General strategies, beRBP is a promising tool for human RBP binding estimation with good prediction accuracy and a broad application scope.

PUF proteins are a conserved group of sequence specific RNA-binding proteins that bind to RNA in a modular fashion. The RNA-binding domain of PUF proteins typically consists of eight clustered Puf repeats. Plant genomes code for large families of PUF proteins that show significant variability in their predicted Puf repeat number, organization, and amino acid sequence. Here we sought to determine whether the observed variability in the RNA-binding domains of four plant PUFs results in a preference for nonclassical PUF RNA target sequences. We report the identification of a novel RNA binding sequence for a nucleolar Arabidopsis PUF protein that contains an atypical RNA-binding domain. The Arabidopsis PUM23 (APUM23) binding sequence was 10 nucleotides in length, contained a centrally located UUGA core element, and had a preferred cytosine at nucleotide position 8. These RNA sequence characteristics differ from those of other PUF proteins, because all natural PUFs studied to date bind to RNAs that contain a conserved UGU sequence at their 5' end and lack specificity for cytosine. Gel mobility shift assays validated the identity of the APUM23 binding sequence and supported the location of 3 of the 10 predicted Puf repeats in APUM23, including the cytosine-binding repeat. The preferred 10-nucleotide sequence bound by APUM23 is present within the 18S rRNA sequence, supporting the known role of APUM23 in 18S rRNA maturation. This work also reveals that APUM23, an ortholog of yeast Nop9, could provide an advanced structural backbone for Puf repeat engineering and target-specific regulation of cellular RNAs.

Keeping Up with RNA-Based Regulation in Bacteria: New Roles for RNA Binding Proteins

Stem cells in regenerative tissues must balance factors promoting self‐renewal with those driving differentiation. Faulty regulation of this balance can lead to tumor growth or loss of regenerative ability. The tractable germline of the nematode C. elegans has allowed the identification of a wealth of key stem cell regulatory factors and remarkably, virtually all are RNA binding proteins (RBP). Despite this insight, a mechanistic understanding of how these RBPs regulate their target mRNAs is lacking. Here, we develop an in vivo method to investigate their mechanisms. Our assay probes how a given RBP affects expression of a reporter mRNA when recruited to its 3′ untranslated region (3′UTR). To perform the assay, an RBP of interest is genetically engineered to contain a small λN peptide. The λN peptide binds a specific RNA hairpin, called boxB, with high affinity. Thus, a reporter mRNA engineered to contain boxB hairpins in its 3′UTR will bind the λN tagged RBP and either increase or decrease expression, depending on the RBP regulatory function. By imaging reporter expression in germline stem cells, we can gain insight into how the RBP regulates target transcripts in its normal biological context. The simple and rapid readout of the assay allows it to be used to probe how an RBP accomplishes its RNA regulation or screen for other factors necessary for RBP function. We have applied this assay to several of the key stem cell RBP regulators to test their functions in vivo. Our results demonstrate the tractability and ease of this method to study RBPs in the C. elegans germline as well as providing mechanistic insights into how the RBPs regulate stem cell fate decisions.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

IA13: RNA regulators and the control of self-renewal

RNA regulation is shifting to the forefront of gene expression in part because it has been difficult to study in the past; but new tools are making it more amenable to exploration on several levels from splicing to export to stability and translation. The field of RNA-protein interactions emerged with the cloning of cDNAs expressing RNA-binding proteins (RBPs) while the enzymes to synthesize cRNA and to map binding sites of RBPs with precision became available and provided important information related to RBP functions in splicing and translational control. In the early 1990s it was assumed that gene expression was solely coordinated at transcription, and that RNA-protein interactions were essentially single events. However, by using in vitro selection of naturally occurring sequences, it was demonstrated that RBPs could be multi-targeted on a larger scale to bind similar sequence elements in the 3′ UTRs of hundreds of brain-derived mRNAs. Later, microarrays allowed investigators to assess the steady-state levels of transcripts and were subsequently used to identify functionally related mRNAs associated with RBPs that govern their localization, coordination and dynamics. Many of the tools used in the discovery of coherent groups of mRNAs coordinated by RBPs in the immune system will be described. Moreover, the use of microarrays and next generation high-throughput sequencing procedures for detecting functional interactions of mRNAs and noncoding RNAs with regulatory RBPs on a genome-wide scale during immune responses will be presented.

Accumulating evidence indicates importance of RNA regulation in cancer. This includes events such as splicing, translation, and regulation of noncoding RNAs, functions which are governed by RNA binding proteins (RBPs). To find which RBPs could be relevant for prostate cancer, we performed systematic screening of RBP expression in clinical prostate cancer. We interrogated four proteome-wide proteomics datasets including tumor samples of primary, castration resistant, and metastatic prostate cancer. We found that, while the majority of RBPs are expressed but not significantly altered during prostate cancer development and progression, expression of several RBPs increases in advanced disease. Interestingly, most of the differentially expressed RBPs are not targets of differential posttranscriptional phosphorylation during disease progression. The RBPs undergoing expression changes have functions in, especially, poly(A)-RNA binding, nucleocytoplasmic transport, and cellular stress responses, suggesting that these may play a role in formation of castration resistance. Pathway analyzes indicate that increased ribosome production and chromatin-related functions of RBPs are also linked to castration resistant and metastatic prostate cancers. We selected a group of differentially expressed RBPs and studied their role in cultured prostate cancer cells. With siRNA screens, several of these were indicated in survival (DDX6, EIF4A3, PABPN1), growth (e.g., EIF5A, HNRNPH2, LRRC47, and NVL), and migration (e.g., NOL3 and SLTM) of prostate cancer cells. Our analyzes further show that RRP9, a U3 small nucleolar protein essential for ribosome formation, undergoes changes at protein level during metastasis in prostate cancer. In this work, we recognized significant molecular alterations in RBP profiles during development and evolution of prostate cancer. Our study further indicates several functionally significant RBPs warranting further investigation for their functions and possible targetability in prostate cancer.

RNA binding proteins (RBPs) act as critical facilitators of spatially regulated gene expression. Muscleblind-like (MBNL) proteins, implicated in myotonic dystrophy and cancer, localize RNAs to myoblast membranes and neurites through unknown mechanisms. We find that MBNL forms motile and anchored granules in neurons and myoblasts, and selectively associates with kinesins Kif1bα and Kif1c through its zinc finger (ZnF) domains. Other RBPs with similar ZnFs associate with these kinesins, implicating a motor-RBP specificity code. MBNL and kinesin perturbation leads to widespread mRNA mis-localization, including depletion of Nucleolin transcripts from neurites. Live cell imaging and fractionation reveal that the unstructured carboxy-terminal tail of MBNL1 allows for anchoring at membranes. An approach, termed RBP Module Recruitment and Imaging (RBP-MRI), reconstitutes kinesin- and membrane-recruitment functions using MBNL-MS2 coat protein fusions. Our findings decouple kinesin association, RNA binding, and membrane anchoring functions of MBNL while establishing general strategies for studying multi-functional, modular domains of RBPs.