PDGFRα signaling regulates Srsf3 transcript binding to affect PI3K signaling and endosomal trafficking.

Signaling through the platelet-derived growth factor receptor alpha (PDGFRa) plays a critical role in craniofacial development, as mutations in PDGFRA are associated with cleft lip/palate in humans and Pdgfra mutant mouse models display varying degrees of facial clefting. Phosphatidylinositol 3-kinase (PI3K)/Akt is the primary effector of PDGFRα signaling during skeletal development in the mouse. We previously demonstrated that Akt phosphorylates the RNA-binding protein serine/arginine-rich splicing factor 3 (Srsf3) downstream of PI3K-mediated PDGFRa signaling in mouse embryonic palatal mesenchyme (MEPM) cells, leading to its nuclear translocation. We further showed that ablation of Srsf3 in the murine neural crest lineage results in severe midline facial clefting, due to defects in proliferation and survival of cranial neural crest cells, and widespread alternative RNA splicing (AS) changes. Here, we sought to determine the molecular mechanisms by which Srsf3 activity is regulated downstream of PDGFRa signaling to control AS of transcripts necessary for craniofacial development. We demonstrated via enhanced UV-crosslinking and immunoprecipitation (eCLIP) of MEPM cells that PDGF-AA stimulation leads to preferential binding of Srsf3 to exons and loss of binding to canonical Srsf3 CA-rich motifs. Through the analysis of complementary RNA-seq data, we showed that Srsf3 activity results in the preferential inclusion of exons with increased GC content and lower intron to exon length ratio. Moreover, we found that the subset of transcripts that are bound by Srsf3 and undergo AS upon PDGFRα signaling commonly encode regulators of PI3K signaling and early endosomal trafficking. Functional validation studies further confirmed that Srsf3 activity downstream of PDGFRα signaling leads to retention of the receptor in early endosomes and increases in downstream PI3K-mediated Akt signaling. Taken together, our findings reveal that growth factor-mediated phosphorylation of an RNA-binding protein underlies gene expression regulation necessary for mammalian craniofacial development.

Systematic analysis of nonsense variants uncovers peptide release rate as a novel modifier of nonsense-mediated mRNA decay efficiency.

Nonsense variants underlie many genetic diseases. The phenotypic impact of nonsense variants is determined by Nonsense-mediated mRNA decay (NMD), which degrades transcripts with premature termination codons (PTCs). NMD activity varies across transcripts and cellular contexts via poorly understood mechanisms. Here, by leveraging human genetic datasets, we uncover that the amino acid preceding the PTC dramatically affects NMD activity in human cells. We find that glycine codons in particular support high levels of NMD and are enriched before PTCs but depleted before normal termination codons (NTCs). Gly-PTC enrichment is most pronounced in human genes that tolerate loss-of-function variants. This suggests a strong biological impact for Gly-PTC in ensuring robust elimination of potentially toxic truncated proteins from non-essential genes. Biochemical assays revealed that the peptide release rate during translation termination is highly dependent on the identity of the amino acid preceding the stop codon. This release rate is the most critical feature determining NMD activity across our massively parallel reporter assays. Together, we conclude that NMD activity is significantly modulated by the "window of opportunity" offered by translation termination kinetics. Integrating the window of opportunity model with the existing framework of NMD would enable more accurate nonsense variant interpretation in the clinic.

In Situ Spatial Reconstruction of Distinct Normal and Pathological Cell Populations Within the Human Adrenal Gland.

The human adrenal gland consists of concentrically organized, functionally distinct regions responsible for hormone production. Dysregulation of adrenocortical cell differentiation alters the proportion and organization of the functional zones of the adrenal cortex leading to disease. Current models of adrenocortical cell differentiation are based on mouse studies, but there are known organizational and functional differences between human and mouse adrenal glands. This study aimed to investigate the centripetal differentiation model in the human adrenal cortex and characterize aldosterone-producing micronodules (APMs) to better understand adrenal diseases such as primary aldosteronism. We applied spatially resolved in situ transcriptomics to human adrenal tissue sections from 2 individuals and identified distinct cell populations and their positional relationships. The results supported the centripetal differentiation model in humans, with cells progressing from the outer capsule to the zona glomerulosa, zona fasciculata, and zona reticularis. Additionally, we characterized 2 APMs in a 72-year-old woman. Comparison with earlier APM transcriptomes indicated a subset of core genes, but also heterogeneity between APMs. The findings contribute to our understanding of normal and pathological cellular differentiation in the human adrenal cortex.

Small-molecule Ro-08-2750 interacts with many RNA-binding proteins and elicits MUSASHI2-independent phenotypes.

RNA-binding proteins (RBPs) are key regulators of gene expression. Small molecules targeting these RBP-RNA interactions are a rapidly emerging class of therapeutics for treating a variety of diseases. Ro-08-2750 (Ro) is a small molecule identified as a competitive inhibitor of Musashi (MSI)-RNA interactions. Here, we show that multiple Ro-dependent cellular phenotypes, specifically adrenocortical steroid production and cell viability, are Musashi-2 (MSI2)-independent. Using an unbiased proteome-wide approach, we discovered Ro broadly interacts with RBPs, many containing RRM domains. To confirm this finding, we leveraged the large-scale ENCODE data to identify a subset of RBPs whose depletion phenocopies Ro inhibition, indicating Ro is a promiscuous inhibitor of multiple RBPs. Consistent with broad disruption of ribonucleoprotein complexes, Ro treatment leads to stress granule formation. This strategy represents a generalizable framework for validating the specificity and identifying targets of RBP inhibitors in a cellular context.

Transcriptomic Response Dynamics of Human Primary and Immortalized Adrenocortical Cells to Steroidogenic Stimuli.

Adrenal steroid hormone production is a dynamic process stimulated by adrenocorticotropic hormone (ACTH) and angiotensin II (AngII). These ligands initialize a rapid and robust gene expression response required for steroidogenesis. Here, we compare the predominant human immortalized cell line model, H295R cell, with primary cultures of adult adrenocortical cells derived from human kidney donors. We performed temporally resolved RNA-seq on primary cells stimulated with either ACTH or AngII at multiple time points. The magnitude of the expression dynamics elicited by ACTH was greater than AngII in primary cells. This is likely due to the larger population of adrenocortical cells that are responsive to ACTH. The dynamics of stimulus-induced expression in H295R cells are mostly recapitulated in primary cells. However, there are some expression responses in primary cells absent in H295R cells. These data are a resource for the endocrine community and will help researchers determine whether H295R is an appropriate model for the specific aspect of steroidogenesis that they are studying.

An Easy, Cost-Effective, and Scalable Method to Deplete Human Ribosomal RNA for RNA-seq.

RNA sequencing (RNA-seq) is a powerful and increasingly prevalent method to characterize and quantify the transcriptome. Ribosomes are extremely abundant, however, and approximately 80% of total RNA is ribosomal RNA (rRNA). Therefore, to detect and quantify less abundant yet biologically important transcripts such as messenger RNA (mRNA) and long noncoding RNAs (lncRNA), it is essential to minimize the rRNA being sequenced. Although commercial methods exist to deplete rRNA from total RNA samples before sequencing, they are expensive and require specific amounts of input RNA, and the most commonly used kit is no longer available as a stand-alone product. Here, we present an optimized rRNA depletion protocol using RNase H and DNA oligonucleotides complementary to human rRNA transcripts. This protocol includes guidelines for DNA oligo preparation, RNA:DNA hybridization, RNase H cleavage and RNA cleanup, and benchmarking of rRNA depletion. The method is flexible because the user can include additional complementary DNA oligos directed against any abundant transcript in their particular system. Furthermore, the performance of this rRNA depletion approach is comparable to or better than that of commercial kits, at a fraction of the cost and across a wide range of input RNA amounts. © 2021 Wiley Periodicals LLC. Basic Protocol: Specific depletion of rRNA transcripts from human total RNA Support Protocol: Preparation of the rRNA depletion DNA oligonucleotide pool.

RNA-binding proteins regulate aldosterone homeostasis in human steroidogenic cells.

Angiotensin II (AngII) stimulates adrenocortical cells to produce aldosterone, a master regulator of blood pressure. Despite extensive characterization of the transcriptional and enzymatic control of adrenocortical steroidogenesis, there are still major gaps in the precise regulation of AII-induced gene expression kinetics. Specifically, we do not know the regulatory contribution of RNA-binding proteins (RBPs) and RNA decay, which can control the timing of stimulus-induced gene expression. To investigate this question, we performed a high-resolution RNA-seq time course of the AngII stimulation response and 4-thiouridine pulse labeling in a steroidogenic human cell line (H295R). We identified twelve temporally distinct gene expression responses that contained mRNA encoding proteins known to be important for various steps of aldosterone production, such as cAMP signaling components and steroidogenic enzymes. AngII response kinetics for many of these mRNAs revealed a coordinated increase in both synthesis and decay. These findings were validated in primary human adrenocortical cells stimulated ex vivo with AngII. Using a candidate screen, we identified a subset of RNA-binding protein and RNA decay factors that activate or repress AngII-stimulated aldosterone production. Among the repressors of aldosterone were BTG2, which promotes deadenylation and global RNA decay. BTG2 was induced in response to AngII stimulation and promoted the repression of mRNAs encoding pro-steroidogenic factors indicating the existence of an incoherent feedforward loop controlling aldosterone homeostasis. These data support a model in which coordinated increases in transcription and decay facilitate the major transcriptomic changes required to implement a pro-steroidogenic expression program that actively resolved to prevent aldosterone overproduction.

ELAVL1 primarily couples mRNA stability with the 3' UTRs of interferon-stimulated genes.

Upon pathogen detection, the innate immune system triggers signaling events leading to upregulation of pro-inflammatory and anti-microbial mRNA transcripts. RNA-binding proteins (RBPs) interact with these critical mRNAs and regulate their fates at the post-transcriptional level. One such RBP is ELAVL1. Although significant progress has been made in understanding how embryonic lethal vision-like protein 1 (ELAVL1) regulates mRNAs, its target repertoire and binding distribution within an immunological context remain poorly understood. We overlap four high-throughput approaches to define its context-dependent targets and determine its regulatory impact during immune activation. ELAVL1 transitions from binding overwhelmingly intronic sites to 3' UTR sites upon immune stimulation of cells, binding previously and newly expressed mRNAs. We find that ELAVL1 mediates the RNA stability of genes that regulate pathways essential to pathogen sensing and cytokine production. Our findings reveal the importance of examining RBP regulatory impact under dynamic transcriptomic events to understand their post-transcriptional regulatory roles within specific biological circuitries.

Towards a deeper annotation of human lncRNAs.

A substantial fraction of the human transcriptome is composed of the so-called long noncoding RNAs (lncRNAs), yet the available catalogs of known lncRNAs are far from complete. Moreover, functional studies of these RNAs are challenged by several factors, such as their tissue-specific expression and functional heterogeneity, resulting in only ca. 1% of them being well characterized. Here, we describe a set of 41,400 novel lncRNAs discovered with RNA-Seq data from 1463 samples encompassing diverse tissues and cell lines. We utilized publicly available transcriptomic and genomic data to provide their characteristics, such as tissue specificity, cellular abundance, polyA status, cellular localization, evolutionary conservation and transcript stability, which allowed us to speculate on their possible biological roles. We also pinpointed 24 novel lncRNAs as candidates for breast cancer biomarkers. The results bring us closer to a comprehensive annotation of human lncRNAs, though vast amounts of further work are needed to validate the predictions and fully decipher their biology. This article is part of a Special Issue entitled: ncRNA in control of gene expression edited by Kotb Abdelmohsen.

Global identification of functional microRNA-mRNA interactions in Drosophila.

MicroRNAs (miRNAs) are key mediators of post-transcriptional gene expression silencing. So far, no comprehensive experimental annotation of functional miRNA target sites exists in Drosophila. Here, we generated a transcriptome-wide in vivo map of miRNA-mRNA interactions in Drosophila melanogaster, making use of single nucleotide resolution in Argonaute1 (AGO1) crosslinking and immunoprecipitation (CLIP) data. Absolute quantification of cellular miRNA levels presents the miRNA pool in Drosophila cell lines to be more diverse than previously reported. Benchmarking two CLIP approaches, we identify a similar predictive potential to unambiguously assign thousands of miRNA-mRNA pairs from AGO1 interaction data at unprecedented depth, achieving higher signal-to-noise ratios than with computational methods alone. Quantitative RNA-seq and sub-codon resolution ribosomal footprinting data upon AGO1 depletion enabled the determination of miRNA-mediated effects on target expression and translation. We thus provide the first comprehensive resource of miRNA target sites and their quantitative functional impact in Drosophila.

Deciphering human ribonucleoprotein regulatory networks.

RNA-binding proteins (RBPs) control and coordinate each stage in the life cycle of RNAs. Although in vivo binding sites of RBPs can now be determined genome-wide, most studies typically focused on individual RBPs. Here, we examined a large compendium of 114 high-quality transcriptome-wide in vivo RBP-RNA cross-linking interaction datasets generated by the same protocol in the same cell line and representing 64 distinct RBPs. Comparative analysis of categories of target RNA binding preference, sequence preference, and transcript region specificity was performed, and identified potential posttranscriptional regulatory modules, i.e. specific combinations of RBPs that bind to specific sets of RNAs and targeted regions. These regulatory modules represented functionally related proteins and exhibited distinct differences in RNA metabolism, expression variance, as well as subcellular localization. This integrative investigation of experimental RBP-RNA interaction evidence and RBP regulatory function in a human cell line will be a valuable resource for understanding the complexity of post-transcriptional regulation.

SSMART: sequence-structure motif identification for RNA-binding proteins.

Motivation: RNA-binding proteins (RBPs) regulate every aspect of RNA metabolism and function. There are hundreds of RBPs encoded in the eukaryotic genomes, and each recognize its RNA targets through a specific mixture of RNA sequence and structure properties. For most RBPs, however, only a primary sequence motif has been determined, while the structure of the binding sites is uncharacterized. Results: We developed SSMART, an RNA motif finder that simultaneously models the primary sequence and the structural properties of the RNA targets sites. The sequence-structure motifs are represented as consensus strings over a degenerate alphabet, extending the IUPAC codes for nucleotides to account for secondary structure preferences. Evaluation on synthetic data showed that SSMART is able to recover both sequence and structure motifs implanted into 3'UTR-like sequences, for various degrees of structured/unstructured binding sites. In addition, we successfully used SSMART on high-throughput in vivo and in vitro data, showing that we not only recover the known sequence motif, but also gain insight into the structural preferences of the RBP. Availability and implementation: SSMART is freely available at https://ohlerlab.mdc-berlin.de/software/SSMART_137/. Supplementary information: Supplementary data are available at Bioinformatics online.

DDX54 regulates transcriptome dynamics during DNA damage response.

The cellular response to genotoxic stress is mediated by a well-characterized network of DNA surveillance pathways. The contribution of post-transcriptional gene regulatory networks to the DNA damage response (DDR) has not been extensively studied. Here, we systematically identified RNA-binding proteins differentially interacting with polyadenylated transcripts upon exposure of human breast carcinoma cells to ionizing radiation (IR). Interestingly, more than 260 proteins, including many nucleolar proteins, showed increased binding to poly(A)+ RNA in IR-exposed cells. The functional analysis of DDX54, a candidate genotoxic stress responsive RNA helicase, revealed that this protein is an immediate-to-early DDR regulator required for the splicing efficacy of its target IR-induced pre-mRNAs. Upon IR exposure, DDX54 acts by increased interaction with a well-defined class of pre-mRNAs that harbor introns with weak acceptor splice sites, as well as by protein-protein contacts within components of U2 snRNP and spliceosomal B complex, resulting in lower intron retention and higher processing rates of its target transcripts. Because DDX54 promotes survival after exposure to IR, its expression and/or mutation rate may impact DDR-related pathologies. Our work indicates the relevance of many uncharacterized RBPs potentially involved in the DDR.

Integrative classification of human coding and noncoding genes through RNA metabolism profiles.

Pervasive transcription of the human genome results in a heterogeneous mix of coding RNAs and long noncoding RNAs (lncRNAs). Only a small fraction of lncRNAs have demonstrated regulatory functions, thus making functional lncRNAs difficult to distinguish from nonfunctional transcriptional byproducts. This difficulty has resulted in numerous competing human lncRNA classifications that are complicated by a steady increase in the number of annotated lncRNAs. To address these challenges, we quantitatively examined transcription, splicing, degradation, localization and translation for coding and noncoding human genes. We observed that annotated lncRNAs had lower synthesis and higher degradation rates than mRNAs and discovered mechanistic differences explaining slower lncRNA splicing. We grouped genes into classes with similar RNA metabolism profiles, containing both mRNAs and lncRNAs to varying extents. These classes exhibited distinct RNA metabolism, different evolutionary patterns and differential sensitivity to cellular RNA-regulatory pathways. Our classification provides an alternative to genomic context-driven annotations of lncRNAs.

Delivery of Therapeutics Targeting the mRNA-Binding Protein HuR Using 3DNA Nanocarriers Suppresses Ovarian Tumor Growth.

Growing evidence shows that cancer cells use mRNA-binding proteins and miRNAs to posttranscriptionally regulate signaling pathways to adapt to harsh tumor microenvironments. In ovarian cancer, cytoplasmic accumulation of mRNA-binding protein HuR (ELAVL1) is associated with poor prognosis. In this study, we observed high HuR expression in ovarian cancer cells compared with ovarian primary cells, providing a rationale for targeting HuR. RNAi-mediated silencing of HuR in ovarian cancer cells significantly decreased cell proliferation and anchorage-independent growth, and impaired migration and invasion. In addition, HuR-depleted human ovarian xenografts were smaller than control tumors. A biodistribution study showed effective tumor-targeting by a novel Cy3-labeled folic acid (FA)-derivatized DNA dendrimer nanocarrier (3DNA). We combined siRNAs against HuR with FA-3DNA and found that systemic administration of the resultant FA-3DNA-siHuR conjugates to ovarian tumor-bearing mice suppressed tumor growth and ascites development, significantly prolonging lifespan. NanoString gene expression analysis identified multiple HuR-regulated genes that function in many essential cellular and molecular pathways, an attractive feature of candidate therapeutic targets. Taken together, these results are the first to demonstrate the versatility of the 3DNA nanocarrier for in vivo-targeted delivery of a cancer therapeutic and support further preclinical investigation of this system adapted to siHuR-targeted therapy for ovarian cancer.

Identifying RBP Targets with RIP-seq.

Throughout their lifetime RNA molecules interact with a variety of RNA-binding proteins (RBPs). RBPs control gene expression by regulating splicing, polyadenylation, editing, transport, stability, and translation of RNA. There are ~1500 RBPs encoded by the human genome and recent studies have detected ~1100 proteins directly interacting with polyadenylated RNA. Identifying the RNAs bound by RBPs will continue to provide important insights into the regulation of gene expression.

Detecting actively translated open reading frames in ribosome profiling data.

RNA-sequencing protocols can quantify gene expression regulation from transcription to protein synthesis. Ribosome profiling (Ribo-seq) maps the positions of translating ribosomes over the entire transcriptome. We have developed RiboTaper (available at https://ohlerlab.mdc-berlin.de/software/), a rigorous statistical approach that identifies translated regions on the basis of the characteristic three-nucleotide periodicity of Ribo-seq data. We used RiboTaper with deep Ribo-seq data from HEK293 cells to derive an extensive map of translation that covered open reading frame (ORF) annotations for more than 11,000 protein-coding genes. We also found distinct ribosomal signatures for several hundred upstream ORFs and ORFs in annotated noncoding genes (ncORFs). Mass spectrometry data confirmed that RiboTaper achieved excellent coverage of the cellular proteome. Although dozens of novel peptide products were validated in this manner, few of the currently annotated long noncoding RNAs appeared to encode stable polypeptides. RiboTaper is a powerful method for comprehensive de novo identification of actively used ORFs from Ribo-seq data.

Identification of the RNA recognition element of the RBPMS family of RNA-binding proteins and their transcriptome-wide mRNA targets.

Recent studies implicated the RNA-binding protein with multiple splicing (RBPMS) family of proteins in oocyte, retinal ganglion cell, heart, and gastrointestinal smooth muscle development. These RNA-binding proteins contain a single RNA recognition motif (RRM), and their targets and molecular function have not yet been identified. We defined transcriptome-wide RNA targets using photoactivatable-ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP) in HEK293 cells, revealing exonic mature and intronic pre-mRNA binding sites, in agreement with the nuclear and cytoplasmic localization of the proteins. Computational and biochemical approaches defined the RNA recognition element (RRE) as a tandem CAC trinucleotide motif separated by a variable spacer region. Similar to other mRNA-binding proteins, RBPMS family of proteins relocalized to cytoplasmic stress granules under oxidative stress conditions suggestive of a support function for mRNA localization in large and/or multinucleated cells where it is preferentially expressed.

Global target mRNA specification and regulation by the RNA-binding protein ZFP36.

BACKGROUND: ZFP36, also known as tristetraprolin or TTP, and ELAVL1, also known as HuR, are two disease-relevant RNA-binding proteins (RBPs) that both interact with AU-rich sequences but have antagonistic roles. While ELAVL1 binding has been profiled in several studies, the precise in vivo binding specificity of ZFP36 has not been investigated on a global scale. We determined ZFP36 binding preferences using cross-linking and immunoprecipitation in human embryonic kidney cells, and examined the combinatorial regulation of AU-rich elements by ZFP36 and ELAVL1. RESULTS: Targets bound and negatively regulated by ZFP36 include transcripts encoding proteins necessary for immune function and cancer, and transcripts encoding other RBPs. Using partial correlation analysis, we were able to quantify the association between ZFP36 binding sites and differential target RNA abundance upon ZFP36 overexpression independent of effects from confounding features. Genes with increased mRNA half-lives in ZFP36 knockout versus wild-type mouse cells were significantly enriched for our human ZFP36 targets. We identified thousands of overlapping ZFP36 and ELAVL1 binding sites, in 1,313 genes, and found that ZFP36 degrades transcripts through specific AU-rich sequences, representing a subset of the U-rich sequences ELAVL1 interacts with to stabilize transcripts. CONCLUSIONS: ZFP36-RNA target specificities in vivo are quantitatively similar to previously reported in vitro binding affinities. ZFP36 and ELAVL1 bind an overlapping spectrum of RNA sequences, yet with differential relative preferences that dictate combinatorial regulatory potential. Our findings and methodology delineate an approach to unravel in vivo combinatorial regulation by RNA-binding proteins.

Integrated detection of natural antisense transcripts using strand-specific RNA sequencing data.

Pairs of RNA molecules transcribed from partially or entirely complementary loci are called cis-natural antisense transcripts (cis-NATs), and they play key roles in the regulation of gene expression in many organisms. A promising experimental tool for profiling sense and antisense transcription is strand-specific RNA sequencing (ssRNA-seq). To identify cis-NATs using ssRNA-seq, we developed a new computational method based on a model comparison framework that incorporates the inherent variable efficiency of generating perfectly strand-specific libraries. Applying the method to new ssRNA-seq data from whole-root and cell-type-specific Arabidopsis libraries confirmed most of the known cis-NAT pairs and identified 918 additional cis-NAT pairs. Newly identified cis-NAT pairs are supported by polyadenylation data, alternative splicing patterns, and RT-PCR validation. We found 209 cis-NAT pairs that have opposite expression levels in neighboring cell types, implying cell-type-specific roles for cis-NATs. By integrating a genome-wide epigenetic profile of Arabidopsis, we identified a unique chromatin signature of cis-NATs, suggesting a connection between cis-NAT transcription and chromatin modification in plants. An analysis of small-RNA sequencing data showed that ∼4% of cis-NAT pairs produce putative cis-NAT-induced siRNAs. Taken together, our data and analyses illustrate the potential for multifaceted regulatory roles of plant cis-NATs.