scholarly journals The RNA-binding landscape of HAX1 protein indicates its involvement in ribosome biogenesis and translation.

2022 ◽  
Anna Balcarak ◽  
Ewelina Macech-Klicka ◽  
Maciej Wakula ◽  
Rafal Tomecki ◽  
Krzysztof Goryca ◽  

HAX1 is a human protein with no known homologues or structural domains, mutations in which cause severe congenital neutropenia through mechanisms that are poorly understood. Previous studies reported RNA-binding capacity of HAX1, but the role of this binding in physiology and pathology remains unexplained. Here we report transcriptome-wide characterization of HAX1 RNA targets using RIP-seq and CRAC, indicating that HAX1 binds transcripts involved in ribosome biogenesis and rRNA processing. Using CRISPR knockouts we find that RNA targets of HAX1 partially overlap with transcripts downregulated in HAX1 KO, implying a role in mRNA stabilization. Gene ontology analysis demonstrated that genes differentially expressed in HAX1 KO (including genes involved in ribosome biogenesis and translation) are also enriched in a subset of genes whose expression correlates with HAX1 expression in four analyzed neoplasms. Functional connection to ribosome biogenesis was also demonstrated by gradient sedimentation ribosome profiles, which revealed differences in the small subunit:monosome ratio in HAX1 WT/KO. We speculate that changes in HAX1 expression may be important for the etiology of HAX1-linked diseases through dysregulation of translation.

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 5056-5056 ◽  
Julia Fremerey ◽  
Pavel Morozov ◽  
Cindy Meyer ◽  
Aitor Garzia ◽  
Marianna Teplova ◽  

Abstract Introduction Nucleolin (NCL) is a multifunctional, proliferation-associated factor that is overexpressed in many cancers and has already been demonstrated to play a profound role in leukemogenesis (Abdelmohsen and Gorospe, 2012; Shen et al., 2014). This can be linked to an increased synthesis of ribosomal RNA (rRNA). Thus, in leukemic cells, high expression levels of NCL contribute to malignant transformation through the increase of rRNA synthesis, which is required to sustain high levels of protein synthesis. Physiologically, NCL is a highly abundant, nucleolar RNA-binding protein that is implicated in the regulation of polymerase I transcription, post-transcriptional gene regulation, and plays a central role in ribosome biogenesis (Srivastava and Pollard, 1999). To further elucidate the exact role of NCL, this study focused on the characterization of the RNA-binding properties and protein-interactions of NCL in the context of ribosome biogenesis. Methods In order to identify transcriptome-wide binding sites and the cellular RNA targets of NCL, PAR-CLIP (photoactivatable-ribonucleoside-enhanced crosslinking and immunoprecipitation) and RIP-Seq (RNA immunoprecipitation sequencing) analyses were carried out in HEK 293 cells. PAR-CLIP is characterized by the incorporation of 4-thiouridine into newly transcribed RNA that causes a T to C conversion in the corresponding cDNA of crosslinked RNA (Hafner et al., 2010). The RNA-binding properties and the interaction of NCL with its identified RNA targets were elucidated by electrophoretic mobility shift assays, isothermal titration calorimetry and size-exclusion chromatography. To further define the role of NCL in ribosome biogenesis and the effect on precursor rRNA levels, siRNA mediated knockdown of NCL was employed followed by RNA sequencing. Furthermore, to characterize the interaction network of NCL on a proteome-wide level, mass-spectrometry was performed. Results This study focuses on the characterization of the RNA-binding properties of NCL and provides the first PAR-CLIP data set of NCL and identifies small nucleolar RNAs (snoRNA) and precursor rRNA as main targets of NCL, both of which were further confirmed by RIP-Seq analysis. Binding sites of NCL were identified in the 5'ETS (external transcribed spacer), after the first cleavage site, in ITS1 and ITS2 (internal transcribed spacer) within the precursor rRNA, indicating that NCL might play a role in the early processing steps of ribosome biogenesis within the nucleolus. Biochemical and structural binding analyses reveal that NCL interacts along the complete precursor region and shows high binding affinity to G/C/U-rich repeat sequences, which is in agreement with the nucleotide composition of the primary rRNA transcript. Moreover, we propose that siRNA mediated knockdown of NCL inhibits polymerase I transcription, which is shown by decreased expression levels of the precursor rRNA transcript. On the proteome-wide level, mass-spectrometry analysis of NCL identified several interaction partners including block of proliferation 1 (BOP1), DEAD-box RNA helicase 18 (DDX18), and 5'-3' exoribonuclease 2 (XRN2) and numerous ribosomal proteins of the small and the large ribosomal subunits including RPS24, RPL11, RPL35A, and RPL36. Conclusion This study provides evidence that NCL is highly associated with the process of ribosome biogenesis on the proteome- and transcriptome-wide level. Therefore, NCL might serve as a promising biochemical target in the context of increased ribosome biogenesis in cancer. Disclosures No relevant conflicts of interest to declare.

2016 ◽  
Vol 113 (42) ◽  
pp. 11967-11972 ◽  
Pan Zhu ◽  
Yuqiu Wang ◽  
Nanxun Qin ◽  
Feng Wang ◽  
Jia Wang ◽  

Ribosome production in eukaryotes requires the complex and precise coordination of several hundred assembly factors, including many small nucleolar RNAs (snoRNAs). However, at present, the distinct role of key snoRNAs in ribosome biogenesis remains poorly understood in higher plants. Here we report that a previously uncharacterized C (RUGAUGA)/D (CUGA) type snoRNA, HIDDEN TREASURE 2 (HID2), acts as an important regulator of ribosome biogenesis through a snoRNA–rRNA interaction. Nucleolus-localized HID2 is actively expressed in Arabidopsis proliferative tissues, whereas defects in HID2 cause a series of developmental defects reminiscent of ribosomal protein mutants. HID2 associates with the precursor 45S rRNA and promotes the efficiency and accuracy of pre-rRNA processing. Intriguingly, disrupting HID2 in Arabidopsis appears to impair the integrity of 27SB, a key pre-rRNA intermediate that generates 25S and 5.8S rRNA and is known to be vital for the synthesis of the 60S large ribosomal subunit and also produces an imbalanced ribosome profile. Finally, we demonstrate that the antisense-box of HID2 is both functionally essential and highly conserved in eukaryotes. Overall, our study reveals the vital and possibly conserved role of a snoRNA in monitoring the efficiency of pre-rRNA processing during ribosome biogenesis.

2020 ◽  
Vol 21 (3) ◽  
pp. 1151 ◽  
Shannon E. Dougherty ◽  
Austin O. Maduka ◽  
Toshifumi Inada ◽  
Gustavo M. Silva

The eukaryotic proteome has to be precisely regulated at multiple levels of gene expression, from transcription, translation, and degradation of RNA and protein to adjust to several cellular conditions. Particularly at the translational level, regulation is controlled by a variety of RNA binding proteins, translation and associated factors, numerous enzymes, and by post-translational modifications (PTM). Ubiquitination, a prominent PTM discovered as the signal for protein degradation, has newly emerged as a modulator of protein synthesis by controlling several processes in translation. Advances in proteomics and cryo-electron microscopy have identified ubiquitin modifications of several ribosomal proteins and provided numerous insights on how this modification affects ribosome structure and function. The variety of pathways and functions of translation controlled by ubiquitin are determined by the various enzymes involved in ubiquitin conjugation and removal, by the ubiquitin chain type used, by the target sites of ubiquitination, and by the physiologic signals triggering its accumulation. Current research is now elucidating multiple ubiquitin-mediated mechanisms of translational control, including ribosome biogenesis, ribosome degradation, ribosome-associated protein quality control (RQC), and redox control of translation by ubiquitin (RTU). This review discusses the central role of ubiquitin in modulating the dynamism of the cellular proteome and explores the molecular aspects responsible for the expanding puzzle of ubiquitin signals and functions in translation.

1991 ◽  
Vol 279 (3) ◽  
pp. 787-792 ◽  
D M Poole ◽  
A J Durrant ◽  
G P Hazlewood ◽  
H J Gilbert

The N-terminal 160 or 267 residues of xylanase A from Pseudomonas fluorescens subsp. cellulosa, containing a non-catalytic cellulose-binding domain (CBD), were fused to the N-terminus of the catalytic domain of endoglucanase E (EGE') from Clostridium thermocellum. A further hybrid enzyme was constructed consisting of the 347 N-terminal residues of xylanase C (XYLC) from P. fluorescens subsp. cellulosa, which also constitutes a CBD, fused to the N-terminus of endoglucanase A (EGA) from Ruminococcus albus. The three hybrid enzymes bound to insoluble cellulose, and could be eluted such that cellulose-binding capacity and catalytic activity were retained. The catalytic properties of the fusion enzymes were similar to EGE' and EGA respectively. Residues 37-347 and 34-347 of XYLC were fused to the C-terminus of EGE' and the 10 amino acids encoded by the multiple cloning sequence of pMTL22p respectively. The two hybrid proteins did not bind cellulose, although residues 39-139 of XYLC were shown previously to constitute a functional CBD. The putative role of the P. fluorescens subsp. cellulosa CBD in cellulase action is discussed.

2021 ◽  
Vol 11 (Suppl_1) ◽  
pp. S21-S21
Andrey Buyan ◽  
Ivan Kulakovskiy ◽  
Sergey Dmitriev

Background: The ribosome is a protein-synthesizing molecular machine composed of four ribosomal RNAs (rRNAs) and dozens of ribosomal proteins. In mammals, the ribosome has a complicated structure with an additional outer layer of rRNA, including large tentacle-like extensions. A number of RNA binding proteins (RBPs) interact with this layer to assist ribosome biogenesis, nuclear export and decay, or to modulate translation. Plenty of methods have been developed in the last decade in order to study such protein-RNA interactions, including RNA pulldown and crosslinking-immunoprecipitation (CLIP) assays. Methods: In the current study, using publicly available data of the enhanced CLIP (eCLIP) experiments for 223 proteins studied in the ENCODE project, we found a number of RBPs that bind rRNAs in human cells. To locate their binding sites in rRNAs, we used a newly developed computational protocol for mapping and evaluation of the eCLIP data with the respect to the repetitive sequences. Results: For two proteins with known ribosomal localization, uS3/RPS3 and uS17/RPS11, the identified sites were in good agreement with structural data, thus validating our approach. Then, we identified rRNA contacts of overall 22 RBPs involved in rRNA processing and ribosome maturation (DDX21, DDX51, DDX52, NIP7, SBDS, UTP18, UTP3, WDR3, and WDR43), translational control during stress (SERBP1, G3BP1, SND1), IRES activity (PCBP1/hnRNPE1), and other translation-related functions. In many cases, the identified proteins interact with the rRNA expansion segments (ES) of the human ribosome pointing to their important role in protein synthesis. Conclusion: Our study identifies a number of RBPs as interacting partners of the human ribosome and sheds light on the role of rRNA expansion segments in translation.

2020 ◽  
Vol 477 (4) ◽  
pp. 773-786
Jana Alexandrova ◽  
David Piñeiro ◽  
Rebekah Jukes-Jones ◽  
Ryan Mordue ◽  
Mark Stoneley ◽  

NF-κB repressing factor (NKRF) was recently identified as an RNA binding protein that together with its associated proteins, the 5′–3′ exonuclease XRN2 and the helicase DHX15, is required to process the precursor ribosomal RNA. XRN2 is a multi-functional ribonuclease that is also involved in processing mRNAs, tRNAs and lncRNAs. The activity and stability of XRN2 are controlled by its binding partners, PAXT-1, CDKN2AIP and CDKN2AIPNL. In each case, these proteins interact with XRN2 via an XRN2 binding domain (XTBD), the structure and mode of action of which is highly conserved. Rather surprisingly, although NKRF interacts directly with XRN2, it was not predicted to contain such a domain, and NKRF's interaction with XRN2 was therefore unexplained. We have identified an alternative upstream AUG start codon within the transcript that encodes NKRF and demonstrate that the full-length form of NKRF contains an XTBD that is conserved across species. Our data suggest that NKRF is tethered in the nucleolus by binding directly to rRNA and that the XTBD in the N-terminal extension of NKRF is essential for the retention of XRN2 in this sub-organelle. Thus, we propose NKRF regulates the early steps of pre-rRNA processing during ribosome biogenesis by controlling the spatial distribution of XRN2 and our data provide further support for the XTBD as an XRN2 interacting motif.

2021 ◽  
Muhammad S Azman ◽  
Martin Dodel ◽  
Federica Capraro ◽  
Rupert Faraway ◽  
Maria Dermit ◽  

Oncogenic RAS signaling reprograms gene expression through both transcriptional and post-transcriptional mechanisms. While transcriptional regulation downstream of RAS is relatively well-characterized, how RAS post-transcriptionally modulates gene expression to promote malignancy is unclear. Using quantitative RNA Interactome Capture analysis, we reveal that oncogenic RAS signaling reshapes the RNA-bound proteomic landscape of cancer cells, with a network of nuclear proteins centered around Nucleolin displaying enhanced RNA-binding activity. We show that Nucleolin is phosphorylated downstream of RAS, which increases its binding to pre-ribosomal-RNA (rRNA), boosts rRNA production, and promotes ribosome biogenesis. This Nucleolin-dependent enhancement of ribosome biogenesis is crucial for RAS-induced cancer cell proliferation, and can be targeted therapeutically to inhibit tumor growth. Our results reveal that oncogenic RAS signaling drives ribosome biogenesis by regulating the RNA-binding activity of Nucleolin, and highlights the crucial role of this process in RAS-mediated tumorigenesis.

BMC Genomics ◽  
2020 ◽  
Vol 21 (1) ◽  
Jochen Bathke ◽  
A. Susann Gauernack ◽  
Oliver Rupp ◽  
Lennart Weber ◽  
Christian Preusser ◽  

Abstract Background The archaeal exosome is an exoribonucleolytic multiprotein complex, which degrades single-stranded RNA in 3′ to 5′ direction phosphorolytically. In a reverse reaction, it can add A-rich tails to the 3′-end of RNA. The catalytic center of the exosome is in the aRrp41 subunit of its hexameric core. Its RNA-binding subunits aRrp4 and aDnaG confer poly(A) preference to the complex. The archaeal exosome was intensely characterized in vitro, but still little is known about its interaction with natural substrates in the cell, particularly because analysis of the transcriptome-wide interaction of an exoribonuclease with RNA is challenging. Results To determine binding sites of the exosome to RNA on a global scale, we performed individual-nucleotide resolution UV crosslinking and immunoprecipitation (iCLIP) analysis with antibodies directed against aRrp4 and aRrp41 of the chrenarchaeon Sulfolobus solfataricus. A relatively high proportion (17–19%) of the obtained cDNA reads could not be mapped to the genome. Instead, they corresponded to adenine-rich RNA tails, which are post-transcriptionally synthesized by the exosome, and to circular RNAs (circRNAs). We identified novel circRNAs corresponding to 5′ parts of two homologous, transposase-related mRNAs. To detect preferred substrates of the exosome, the iCLIP reads were compared to the transcript abundance using RNA-Seq data. Among the strongly enriched exosome substrates were RNAs antisense to tRNAs, overlapping 3′-UTRs and RNAs containing poly(A) stretches. The majority of the read counts and crosslink sites mapped in mRNAs. Furthermore, unexpected crosslink sites clustering at 5′-ends of RNAs was detected. Conclusions In this study, RNA targets of an exoribonuclease were analyzed by iCLIP. The data documents the role of the archaeal exosome as an exoribonuclease and RNA-tailing enzyme interacting with all RNA classes, and underlines its role in mRNA turnover, which is important for adaptation of prokaryotic cells to changing environmental conditions. The clustering of crosslink sites near 5′-ends of genes suggests simultaneous binding of both RNA ends by the S. solfataricus exosome. This may serve to prevent translation of mRNAs dedicated to degradation in 3′-5′ direction.

2002 ◽  
Vol 22 (12) ◽  
pp. 4101-4112 ◽  
Nenad Tomasevic ◽  
Brenda A. Peculis

ABSTRACT U8 snoRNA plays a unique role in ribosome biogenesis: it is the only snoRNA essential for maturation of the large ribosomal subunit RNAs, 5.8S and 28S. To learn the mechanisms behind the in vivo role of U8 snoRNA, we have purified to near homogeneity and characterized a set of proteins responsible for the formation of a specific U8 RNA-binding complex. This 75-kDa complex is stable in the absence of added RNA and binds U8 with high specificity, requiring the conserved octamer sequence present in all U8 homologues. At least two proteins in this complex can be cross-linked directly to U8 RNA. We have identified the proteins as Xenopus homologues of the LSm (like Sm) proteins, which were previously reported to be involved in cytoplasmic degradation of mRNA and nuclear stabilization of U6 snRNA. We have identified LSm2, -3, -4, -6, -7, and -8 in our purified complex and found that this complex associates with U8 RNA in vivo. This purified complex can bind U6 snRNA in vitro but does not bind U3 or U14 snoRNA in vitro, demonstrating that the LSm complex specifically recognizes U8 RNA.

Biology ◽  
2021 ◽  
Vol 10 (9) ◽  
pp. 939
Michela Dell’Orco ◽  
Amir Elyaderani ◽  
Annika Vannan ◽  
Shobana Sekar ◽  
Gregory Powell ◽  

The RNA-binding protein HuD (a.k.a., ELAVL4) is involved in neuronal development and synaptic plasticity mechanisms, including addiction-related processes such as cocaine conditioned-place preference (CPP) and food reward. The most studied function of this protein is mRNA stabilization; however, we have recently shown that HuD also regulates the levels of circular RNAs (circRNAs) in neurons. To examine the role of HuD in the control of coding and non-coding RNA networks associated with substance use, we identified sets of differentially expressed mRNAs, circRNAs and miRNAs in the striatum of HuD knockout (KO) mice. Our findings indicate that significantly downregulated mRNAs are enriched in biological pathways related to cell morphology and behavior. Furthermore, deletion of HuD altered the levels of 15 miRNAs associated with drug seeking. Using these sets of data, we predicted that a large number of upregulated miRNAs form competing endogenous RNA (ceRNA) networks with circRNAs and mRNAs associated with the neuronal development and synaptic plasticity proteins LSAMP and MARK3. Additionally, several downregulated miRNAs form ceRNA networks with mRNAs and circRNAs from MEF2D, PIK3R3, PTRPM and other neuronal proteins. Together, our results indicate that HuD regulates ceRNA networks controlling the levels of mRNAs associated with neuronal differentiation and synaptic physiology.

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