RAVER1 hinders lethal EMT and modulates miR/RISC activity by the control of alternative splicing.

The RAVER1 protein serves as a co-factor in guiding the polypyrimidine tract-binding protein (PTBP)-dependent control of alternative splicing (AS). Whether RAVER1 solely acts in concert with PTBPs and how it affects cancer cell fate remained elusive. Here, we provide the first comprehensive investigation of RAVER1-controlled AS in cancer cell models. This reveals a pro-oncogenic role of RAVER1 in modulating tumor growth and epithelial-mesenchymal-transition (EMT). Splicing analyses and protein-association studies indicate that RAVER1 guides AS in association with other splicing regulators, including PTBPs and SRSFs. In cancer cells, one major function of RAVER1 is the stimulation of proliferation and restriction of apoptosis. This involves the modulation of AS events within the miR/RISC pathway. Disturbance of RAVER1 impairs miR/RISC activity resulting in severely deregulated gene expression, which promotes lethal TGFB-driven EMT. Among others, RAVER1-modulated splicing events affect the insertion of protein interaction modules in factors guiding miR/RISC-dependent gene silencing. Most prominently, in all three human TNRC6 proteins, RAVER1 controls AS of GW-enriched motifs, which are essential for AGO2-binding and the formation of active miR/RISC complexes. We propose, that RAVER1 is a key modulator of AS events in the miR/RISC pathway ensuring proper abundance and composition of miR/RISC effectors. This ensures balanced expression of TGFB signaling effectors and limits TGFB induced lethal EMT.

Rbfox1 controls alternative splicing of focal adhesion genes in cardiac muscle cells.

Alternative splicing is one of the major cellular processes that determine the tissue-specific expression of protein variants. However, it remains challenging to identify physiologically relevant and tissue-selective proteins that are generated by alternative splicing. Hence, we investigated the target spectrum of the splicing factor Rbfox1 in the cardiac muscle context in more detail. By using a combination of in silico target prediction and in cell validation, we identified several focal adhesion proteins as alternative splicing targets of Rbfox1. We focused on the alternative splicing patterns of Vinculin (Metavinculin isoform) and Paxillin (extended Paxillin isoform) and identified both as potential Rbfox1 targets. Minigene analyses suggested that both isoforms are promoted by Rbfox1 due to binding in the introns. Focal adhesions play an important role in the cardiac muscle context, since they mainly influence cell shape, cytoskeletal organization, and cell-matrix association. Our data confirmed that depletion of Rbfox1 changed cardiomyoblast morphology, cytoskeletal organization, and multinuclearity after differentiation, which might be due to changes in alternative splicing of focal adhesion proteins. Hence, our results indicate that Rbfox1 promotes alternative splicing of focal adhesion genes in cardiac muscle cells, which might contribute to heart disease progression, where downregulation of Rbfox1 is frequently observed.

The pro-sequence of parathyroid hormone prevents premature amyloid fibril formation.

The parathyroid hormone (PTH) regulates the calcium and phosphate level in blood after secretion from parathyroid chief cells. The pre- and pro-sequences of precursor preproPTH get cleaved during PTH maturation. In secretory granules, PTH forms functional amyloids. Using thioflavin T fibrillation assays, circular dichroism, NMR spectroscopy, and cellular cAMP activation, we show that the pro-sequence prevents premature fibrillation by impairing primary nucleation because of Coulomb repulsion of positively charged residues. Under seeding or high salt conditions or in the presence of heparin at pH 5.5, proPTH fibril formation is delayed, but the monomer release properties are conserved. ProPTH can still activate in cellulo PTH receptor 1 but with impaired potency. These findings give some perspectives on medical applications of PTH in hormone therapy.

Lighting up Nobel Prize-winning studies with protein intrinsic disorder.

Intrinsically disordered proteins and regions (IDPs and IDRs) and their importance in biology are becoming increasingly recognized in biology, biochemistry, molecular biology and chemistry textbooks, as well as in current protein science and structural biology curricula. We argue that the sequence → dynamic conformational ensemble → function principle is of equal importance as the classical sequence → structure → function paradigm. To highlight this point, we describe the IDPs and/or IDRs behind the discoveries associated with 17 Nobel Prizes, 11 in Physiology or Medicine and 6 in Chemistry. The Nobel Laureates themselves did not always mention that the proteins underlying the phenomena investigated in their award-winning studies are in fact IDPs or contain IDRs. In several cases, IDP- or IDR-based molecular functions have been elucidated, while in other instances, it is recognized that the respective protein(s) contain IDRs, but the specific IDR-based molecular functions have yet to be determined. To highlight the importance of IDPs and IDRs as general principle in biology, we present here illustrative examples of IDPs/IDRs in Nobel Prize-winning mechanisms and processes.

Small but Mighty-The Emerging Role of snoRNAs in Hematological Malignancies.

Over recent years, the long known class of small nucleolar RNAs (snoRNAs) have gained interest among the scientific community, especially in the clinical context. The main molecular role of this interesting family of non-coding RNAs is to serve as scaffolding RNAs to mediate site-specific RNA modification of ribosomal RNAs (rRNAs) and small nuclear RNAs (snRNAs). With the development of new sequencing techniques and sophisticated analysis pipelines, new members of the snoRNA family were identified and global expression patterns in disease backgrounds could be determined. We will herein shed light on the current research progress in snoRNA biology and their clinical role by influencing disease outcome in hematological diseases. Astonishingly, in recent studies snoRNAs emerged as potent biomarkers in a variety of these clinical setups, which is also highlighted by the frequent deregulation of snoRNA levels in the hema-oncological context. However, research is only starting to reveal how snoRNAs might influence cellular functions and the connected disease hallmarks in hematological malignancies.

Identification and initial characterization of POLIII-driven transcripts by msRNA-sequencing.

Non-coding RNAs (ncRNAs) are powerful regulators of gene expression but medium-sized (50-300 nts in length) ncRNAs (msRNAs) are barely picked-up precisely by RNA-sequencing. Here we describe msRNA-sequencing (msRNAseq), a modified protocol that associated with a computational analyses pipeline identified about ~1800 msRNA loci, including over 300 putatively novel msRNAs, in human and murine cells. We focused on the identification and initial characterization of three POLIII-derived transcripts. The validation of these uncharacterized msRNAs identified an ncRNA in antisense orientation from the POLR3E locus transcribed by POLIII. This msRNA, termed POLAR (POLR3E Antisense RNA), has a strikingly short half-life, localizes to paraspeckles (PSPs) and associates with PSP-associated proteins indicating that msRNAseq identifies functional msRNAs. Thus, our analyses will pave the way for analysing the roles of msRNAs in cells, development and diseases.

NOP10 predicts lung cancer prognosis and its associated small nucleolar RNAs drive proliferation and migration.

Non-small cell lung cancer (NSCLC) is the leading cause of cancer death worldwide underlining the urgent need for new biomarkers and therapeutic targets for this disease. Long noncoding RNAs are critical players in NSCLC but the role of small RNA species is not well understood. In the present study, we investigated the role of H/ACA box small nucleolar RNAs (snoRNAs) and snoRNA-bound ribonucleoproteins (snoRNPs) in the tumorigenesis of NSCLC. H/ACA box snoRNPs including the NOP10 core protein were highly expressed in NSCLC. High levels of either NOP10 mRNA or protein were associated with poor prognosis in NSCLC patients. Loss of NOP10 and subsequent reduction of H/ACA box snoRNAs and rRNA pseudouridylation inhibited lung cancer cell growth, colony formation, migration, and invasion. A focused CRISPR/Cas9 snoRNA knockout screen revealed that genomic deletion of SNORA65, SNORA7A, and SNORA7B reduced proliferation of lung cancer cells. In line, high levels of SNORA65, SNORA7A, and SNORA7B were observed in primary lung cancer specimens with associated changes in rRNA pseudouridylation. Knockdown of either SNORA65 or SNORA7A/B inhibited growth and colony formation of NSCLC cell lines. Our data indicate that specific H/ACA box snoRNAs and snoRNA-associated proteins such as NOP10 have an oncogenic role in NSCLC providing new potential biomarkers and therapeutic targets for the disease.

RNA Sequencing of Collecting Duct Renal Cell Carcinoma Suggests an Interaction between miRNA and Target Genes and a Predominance of Deregulated Solute Carrier Genes.

Collecting duct carcinoma (CDC) is a rare renal cell carcinoma subtype with a very poor prognosis. There have been only a few studies on gene expression analysis in CDCs. We compared the gene expression profiles of two CDC cases with those of eight normal tissues of renal cell carcinoma patients. At a threshold of |log2fold-change| ≥ 1, 3349 genes were upregulated and 1947 genes were downregulated in CDCs compared to the normal samples. Pathway analysis of the deregulated genes revealed that cancer pathways and cell cycle pathways were most prominent in CDCs. The most upregulated gene was keratin 17, and the most downregulated gene was cubilin. Among the most downregulated genes were four solute carrier genes (SLC3A1, SLC9A3, SLC26A7, and SLC47A1). The strongest negative correlations between miRNAs and mRNAs were found between the downregulated miR-374b-5p and its upregulated target genes HIST1H3B, HK2, and SLC7A11 and between upregulated miR-26b-5p and its downregulated target genes PPARGC1A, ALDH6A1, and MARC2. An upregulation of HK2 and a downregulation of PPARGC1A, ALDH6A1, and MARC2 were observed at the protein level. Survival analysis of the cancer genome atlas (TCGA) dataset showed for the first time that low gene expression of MARC2, cubilin, and SLC47A1 and high gene expression of KRT17 are associated with poor overall survival in clear cell renal cell carcinoma patients. Altogether, we identified dysregulated protein-coding genes, potential miRNA-target interactions, and prognostic markers that could be associated with CDC.

POLIII-derived non-coding RNAs acting as scaffolds and decoys.

A large variety of eukaryotic small structured POLIII-derived non-coding RNAs (ncRNAs) have been described in the past. However, for only few, e.g. 7SL and H1/MRP families, cellular functions are well understood. For the vast majority of these transcripts, cellular functions remain unknown. Recent findings on the role of Y RNAs and other POLIII-derived ncRNAs suggest an evolutionarily conserved function of these ncRNAs in the assembly and function of ribonucleoprotein complexes (RNPs). These RNPs provide cellular `machineries', which are essential for guiding the fate and function of a variety of RNAs. In this review, we summarize current knowledge on the role of POLIII-derived ncRNAs in the assembly and function of RNPs. We propose that these ncRNAs serve as scaffolding factors that `chaperone' RNA-binding proteins (RBPs) to form functional RNPs. In addition or associated with this role, some small ncRNAs act as molecular decoys impairing the RBP-guided control of RNA fate by competing with other RNA substrates. This suggests that POLIII-derived ncRNAs serve essential and conserved roles in the assembly of larger RNPs and thus the control of gene expression by indirectly guiding the fate of mRNAs and lncRNAs.

HuD Is a Neural Translation Enhancer Acting on mTORC1-Responsive Genes and Counteracted by the Y3 Small Non-coding RNA.

The RNA-binding protein HuD promotes neurogenesis and favors recovery from peripheral axon injury. HuD interacts with many mRNAs, altering both stability and translation efficiency. We generated a nucleotide resolution map of the HuD RNA interactome in motor neuron-like cells, identifying HuD target sites in 1,304 mRNAs, almost exclusively in the 3' UTR. HuD binds many mRNAs encoding mTORC1-responsive ribosomal proteins and translation factors. Altered HuD expression correlates with the translation efficiency of these mRNAs and overall protein synthesis, in a mTORC1-independent fashion. The predominant HuD target is the abundant, small non-coding RNA Y3, amounting to 70% of the HuD interaction signal. Y3 functions as a molecular sponge for HuD, dynamically limiting its recruitment to polysomes and its activity as a translation and neuron differentiation enhancer. These findings uncover an alternative route to the mTORC1 pathway for translational control in motor neurons that is tunable by a small non-coding RNA.

Identification of a novel lncRNA induced by the nephrotoxin ochratoxin A and expressed in human renal tumor tissue.

Long non-coding RNAs represent a fraction of the transcriptome that is being increasingly recognized. For most of them no function has been allocated so far. Here, we describe the nature and function of a novel non-protein-coding transcript, named WISP1-AS1, discovered in human renal proximal tubule cells exposed to the carcinogenic nephrotoxin ochratoxin A. WISP1-AS1 overlaps parts of the fourth intron and fifth exon of the Wnt1-inducible signaling pathway protein 1 (WISP1) gene. The transcript is 2922 nucleotides long, transcribed in antisense direction and predominantly localized in the nucleus. WISP1-AS1 is expressed in all 20 samples of a human tissue RNA panel with the highest expression levels detected in uterus, kidney and adrenal gland. Its expression was confirmed in primary tissues of human kidneys. In addition, WISP1-AS1 is expressed at higher levels in renal cell carcinoma (RCC) cell lines compared to primary proximal tubule cells as well as in RCC lesions than in the adjacent healthy control tissue from the same patient. Using specific gapmer antisense oligonucleotides to prevent its upregulation, we show that WISP1-AS1 (1) does not influence the mRNA expression of WISP1, (2) affects transcriptional regulation by Egr-1 and E2F as revealed by RNA-sequencing, enrichment analysis and reporter assays, and (3) modulates the apoptosis-necrosis balance. In summary, WISP1-AS1 is a novel lncRNA with modulatory transcriptional function and the potential to alter the cellular phenotype in situations of stress or oncogenic transformation. However, its precise mode of action and impact on cellular functions require further investigations.

AML1-ETO requires enhanced C/D box snoRNA/RNP formation to induce self-renewal and leukaemia.

Leukaemogenesis requires enhanced self-renewal, which is induced by oncogenes. The underlying molecular mechanisms remain incompletely understood. Here, we identified C/D box snoRNAs and rRNA 2'-O-methylation as critical determinants of leukaemic stem cell activity. Leukaemogenesis by AML1-ETO required expression of the groucho-related amino-terminal enhancer of split (AES). AES functioned by inducing snoRNA/RNP formation via interaction with the RNA helicase DDX21. Similarly, global loss of C/D box snoRNAs with concomitant loss of rRNA 2'-O-methylation resulted in decreased leukaemia self-renewal potential. Genomic deletion of either C/D box snoRNA SNORD14D or SNORD35A suppressed clonogenic potential of leukaemia cells in vitro and delayed leukaemogenesis in vivo. We further showed that AML1-ETO9a, MYC and MLL-AF9 all enhanced snoRNA formation. Expression levels of C/D box snoRNAs in AML patients correlated closely with in vivo frequency of leukaemic stem cells. Collectively, these findings indicate that induction of C/D box snoRNA/RNP function constitutes an important pathway in leukaemogenesis.

Non-coding RNAs, the cutting edge of histone messages.

In metazoan the 3'-end processing of histone mRNAs is a conserved process involving the concerted action of many protein factors and the non-coding U7 snRNA. Recently, we identified that the processing of histone pre-mRNAs is promoted by an additional ncRNA, the Y3-derived Y3** RNA. U7 modulates the association of the U7 snRNP whereas Y3** promotes recruitment of CPSF (cleavage and polyadenylation specific factor) proteins to nascent histone transcripts at histone locus bodies (HLBs) in mammals. This enhances the 3'-end cleavage of nascent histone pre-mRNAs and modulates HLB assembly. Here we discuss new insights in the role of ncRNAs in the spatiotemporal control of histone synthesis. We propose that ncRNAs scaffold the formation of functional protein-RNA complexes and their sequential deposition on nascent histone pre-mRNAs at HLBs. These findings add to the multiple roles of ncRNAs in controlling gene expression and may provide new avenues for targeting histone synthesis in cancer.

The Y3** ncRNA promotes the 3' end processing of histone mRNAs.

We demonstrate that the Y3/Y3** noncoding RNAs (ncRNAs) bind to the CPSF (cleavage and polyadenylation specificity factor) and that Y3** associates with the 3' untranslated region (UTR) of histone pre-mRNAs. The depletion of Y3** impairs the 3' end processing of histone pre-mRNAs as well as the formation and protein dynamics of histone locus bodies (HLBs), the site of histone mRNA synthesis and processing. HLB morphology is also disturbed by knockdown of the CPSF but not the U7-snRNP components. In conclusion, we propose that the Y3** ncRNA promotes the 3' end processing of histone pre-mRNAs by enhancing the recruitment of the CPSF to histone pre-mRNAs at HLBs.

Subcellular localization and RNP formation of IGF2BPs (IGF2 mRNA-binding proteins) is modulated by distinct RNA-binding domains.

The IGF2 mRNA-binding protein family (IGF2BPs) directs the cytoplasmic fate of various target mRNAs and controls essential cellular functions. The three IGF2BP paralogues expressed in mammals comprise two RNA-recognition motifs (RRM) as well as four KH domains. How these domains direct IGF2BP paralogue-dependent protein function remains largely elusive. In this study, we analyze the role of KH domains in IGF2BPs by the mutational GXXG-GEEG conversion of single KH domain loops in the context of full-length polypeptides. These analyses reveal that all four KH domains of IGF2BP1 and IGF2BP2 are essentially involved in RNA-binding in vitro and the cellular association with RNA-binding proteins (RBPs). Moreover the KH domains prevent the nuclear accumulation of these two paralogues and facilitate their recruitment to stress granules. The role of KH domains appears less pronounced in IGF2BP3, because GxxG-GEEG conversion in all four KH domains only modestly affects RNA-binding, subcellular localization and RNA-dependent protein association of this paralogue. These findings indicate paralogue-dependent RNA-binding properties of IGF2BPs which likely direct distinct cellular functions. Our findings suggest that IGF2BPs contact target RNAs via all four KH domains. This implies significant structural constraints, which presumably allow the formation of exceedingly stable protein-RNA complexes.

Insulin-like growth factor 2 mRNA-binding proteins (IGF2BPs): post-transcriptional drivers of cancer progression?

The insulin-like growth factor-2 mRNA-binding proteins 1, 2, and 3 (IGF2BP1, IGF2BP2, IGF2BP3) belong to a conserved family of RNA-binding, oncofetal proteins. Several studies have shown that these proteins act in various important aspects of cell function, such as cell polarization, migration, morphology, metabolism, proliferation and differentiation. In this review, we discuss the IGF2BP family's role in cancer biology and how this correlates with their proposed functions during embryogenesis. IGF2BPs are mainly expressed in the embryo, in contrast with comparatively lower or negotiable levels in adult tissues. IGF2BP1 and IGF2BP3 have been found to be re-expressed in several aggressive cancer types. Control of IGF2BPs' expression is not well understood; however, let-7 microRNAs, ß-catenin (CTNNB1) and MYC have been proposed to be involved in their regulation. In contrast to many other RNA-binding proteins, IGF2BPs are almost exclusively observed in the cytoplasm where they associate with target mRNAs in cytoplasmic ribonucleoprotein complexes (mRNPs). During development, IGF2BPs are required for proper nerve cell migration and morphological development, presumably involving the control of cytoskeletal remodeling and dynamics, respectively. Likewise, IGF2BPs modulate cell polarization, adhesion and migration in tumor-derived cells. Moreover, they are highly associated with cancer metastasis and the expression of oncogenic factors (KRAS, MYC and MDR1). However, a pro-metastatic role of IGF2BPs remains controversial due to the lack of 'classical' in vivo studies. Nonetheless, IGF2BPs could provide valuable targets in cancer treatment with many of their in vivo roles to be fully elucidated.

Why YRNAs? About Versatile RNAs and Their Functions.

Y RNAs constitute a family of highly conserved small noncoding RNAs (in humans: 83-112 nt; Y1, Y3, Y4 and Y5). They are transcribed from individual genes by RNA-polymerase III and fold into conserved stem-loop-structures. Although discovered 30 years ago, insights into the cellular and physiological role of Y RNAs remains incomplete. In this review, we will discuss knowledge on the structural properties, associated proteins and discuss proposed functions of Y RNAs. We suggest Y RNAs to be an integral part of ribonucleoprotein networks within cells and could therefore have substantial influence on many different cellular processes. Putative functions of Y RNAs include small RNA quality control, DNA replication, regulation of the cellular stress response and proliferation. This suggests Y RNAs as essential regulators of cell fate and indicates future avenues of research, which will provide novel insights into the role of small noncoding RNAs in gene expression.

IGF2BP1 promotes cell migration by regulating MK5 and PTEN signaling.

In primary neurons, the oncofetal RNA-binding protein IGF2BP1 (IGF2 mRNA-binding protein 1) controls spatially restricted ß-actin (ACTB) mRNA translation and modulates growth cone guidance. In cultured tumor-derived cells, IGF2BP1 was shown to regulate the formation of lamellipodia and invadopodia. However, how and via which target mRNAs IGF2BP1 controls the motility of tumor-derived cells has remained elusive. In this study, we reveal that IGF2BP1 promotes the velocity and directionality of tumor-derived cell migration by determining the cytoplasmic fate of two novel target mRNAs: MAPK4 and PTEN. Inhibition of MAPK4 mRNA translation by IGF2BP1 antagonizes MK5 activation and prevents phosphorylation of HSP27, which sequesters actin monomers available for F-actin polymerization. Consequently, HSP27-ACTB association is reduced, mobilizing cellular G-actin for polymerization in order to promote the velocity of cell migration. At the same time, stabilization of the PTEN mRNA by IGF2BP1 enhances PTEN expression and antagonizes PIP(3)-directed signaling. This enforces the directionality of cell migration in a RAC1-dependent manner by preventing additional lamellipodia from forming and sustaining cell polarization intrinsically. IGF2BP1 thus promotes the velocity and persistence of tumor cell migration by controlling the expression of signaling proteins. This fine-tunes and connects intracellular signaling networks in order to enhance actin dynamics and cell polarization.

Near-infrared (NIR) dye-labeled RNAs identify binding of ZBP1 to the noncoding Y3-RNA.

The analysis of protein-RNA association in vitro commonly involves radiolabeled in vitro transcribed RNAs. Nucleotides labeled with near-infrared (NIR) dyes provide promising alternatives for studying protein-RNA binding in vitro. However, it remained elusive whether random labeling of RNA probes by NIR dyes interferes with protein binding. Here, we demonstrate that infrared scanning allows the detection of randomly NIR-labeled RNA probes in the low femtomole range. The analyses of eight distinct protein-RNA complexes by electrophoretic mobility shift assay, filter binding, or UV crosslinking revealed that protein binding specificity remains unaffected by random NIR labeling. Accordingly, NIR probes allowed the rapid identification of the short noncoding Y3-RNA as a novel RNA target of ZBP1 (zipcode binding protein). Whereas binding of ZBP1 to the ACTB-zipcode and Y3 was exclusive, the protein formed a trimeric complex with the La protein and Y3. This was dissociated in the presence of Y5 RNA, resulting in the formation of ZBP1/Y3 and La/Y5 complexes. Hence, ZBP1 apparently resides in at least two distinct cellular RNPs: mRNA-containing mRNPs or Y3-containing yRNPs. In conclusion, our findings indicate that randomly labeled NIR probes provide a powerful tool for the rapid and sensitive analysis of protein-RNA binding in vitro. In contrast to radiolabeled RNAs, NIR probes remain stable for months, do not pose any safety considerations, and enable the significantly expedited analysis of experimental data due to fast read technologies available. The most prominent advantage of probes labeled by NIR dyes is the option to color-code distinct transcripts, allowing the unbiased identification of distinct protein-RNA complexes in one sample.

Control of c-myc mRNA stability by IGF2BP1-associated cytoplasmic RNPs.

The RNA-binding protein IGF2BP1 (IGF-II mRNA binding protein 1) stabilizes the c-myc RNA by associating with the Coding Region instability Determinant (CRD). If and how other proteins cooperate with IGF2BP1 in promoting stabilization of the c-myc mRNA via the CRD remained elusive. Here, we identify various RNA-binding proteins that associate with IGF2BP1 in an RNA-dependent fashion. Four of these proteins (HNRNPU, SYNCRIP, YBX1, and DHX9) were essential to ensure stabilization of the c-myc mRNA via the CRD. These factors associate with IGF2BP1 in a CRD-dependent manner, co-distribute with IGF2BP1 in non-polysomal fractions comprising c-myc mRNA, and colocalize with IGF2BP1 in the cytoplasm. A selective shift of relative c-myc mRNA levels to the polysomal fraction is observed upon IGF2BP1 knockdown. These findings suggest that IGF2BP1 in complex with at least four proteins promotes CRD-mediated mRNA stabilization. Complex formation at the CRD presumably limits the transfer of c-myc mRNA to the polysomal fraction and subsequent translation-coupled decay.