Structure and regulation of ZCCHC4 in m6A-methylation of 28S rRNA

Abstract N6-methyladenosine (m6A) modification provides an important epitranscriptomic mechanism that critically regulates RNA metabolism and function. However, how m6A writers attain substrate specificities remains unclear. We report the 3.1 Å-resolution crystal structure of human CCHC zinc finger-containing protein ZCCHC4, a 28S rRNA-specific m6A methyltransferase, bound to S-adenosyl-L-homocysteine. The methyltransferase (MTase) domain of ZCCHC4 is packed against N-terminal GRF-type and C2H2 zinc finger domains and a C-terminal CCHC domain, creating an integrated RNA-binding surface. Strikingly, the MTase domain adopts an autoinhibitory conformation, with a self-occluded catalytic site and a fully-closed cofactor pocket. Mutational and enzymatic analyses further substantiate the molecular basis for ZCCHC4-RNA recognition and a role of the stem-loop structure within substrate in governing the substrate specificity. Overall, this study unveils unique structural and enzymatic characteristics of ZCCHC4, distinctive from what was seen with the METTL family of m6A writers, providing the mechanistic basis for ZCCHC4 modulation of m6A RNA methylation.

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The human methyltransferase ZCCHC4 catalyses N6-methyladenosine modification of 28S ribosomal RNA

Nucleic Acids Research ◽

10.1093/nar/gkz1147 ◽

2019 ◽

Vol 48 (2) ◽

pp. 830-846 ◽

Cited By ~ 6

Author(s):

Rita Pinto ◽

Cathrine B Vågbø ◽

Magnus E Jakobsson ◽

Yeji Kim ◽

Marijke P Baltissen ◽

...

Keyword(s):

Ribosomal Rna ◽

Rna Structure ◽

Mrna Translation ◽

28S Rrna ◽

Loop Structure ◽

Stem Loop ◽

Stem Loop Structure ◽

Reversible Modification ◽

And Shifts ◽

And Function

Abstract RNA methylations are essential both for RNA structure and function, and are introduced by a number of distinct methyltransferases (MTases). In recent years, N6-methyladenosine (m6A) modification of eukaryotic mRNA has been subject to intense studies, and it has been demonstrated that m6A is a reversible modification that regulates several aspects of mRNA function. However, m6A is also found in other RNAs, such as mammalian 18S and 28S ribosomal RNAs (rRNAs), but the responsible MTases have remained elusive. 28S rRNA carries a single m6A modification, found at position A4220 (alternatively referred to as A4190) within a stem–loop structure, and here we show that the MTase ZCCHC4 is the enzyme responsible for introducing this modification. Accordingly, we found that ZCCHC4 localises to nucleoli, the site of ribosome assembly, and that proteins involved in RNA metabolism are overrepresented in the ZCCHC4 interactome. Interestingly, the absence of m6A4220 perturbs codon-specific translation dynamics and shifts gene expression at the translational level. In summary, we establish ZCCHC4 as the enzyme responsible for m6A modification of human 28S rRNA, and demonstrate its functional significance in mRNA translation.

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Terminal uridyltransferase 7 regulates TLR4-triggered inflammation by controlling Regnase-1 mRNA uridylation and degradation

Nature Communications ◽

10.1038/s41467-021-24177-7 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Chia-Ching Lin ◽

Yi-Ru Shen ◽

Chi-Chih Chang ◽

Xiang-Yi Guo ◽

Yun-Yun Young ◽

...

Keyword(s):

Posttranscriptional Regulation ◽

Inflammatory Responses ◽

Rna Stability ◽

Innate Immune ◽

Stem Loop ◽

Stem Loop Structure ◽

Tlr4 Activation ◽

Harmful Effects ◽

Different Levels

AbstractDifferent levels of regulatory mechanisms, including posttranscriptional regulation, are needed to elaborately regulate inflammatory responses to prevent harmful effects. Terminal uridyltransferase 7 (TUT7) controls RNA stability by adding uridines to its 3′ ends, but its function in innate immune response remains obscure. Here we reveal that TLR4 activation induces TUT7, which in turn selectively regulates the production of a subset of cytokines, including Interleukin 6 (IL-6). TUT7 regulates IL-6 expression by controlling ribonuclease Regnase-1 mRNA (encoded by Zc3h12a gene) stability. Mechanistically, TLR4 activation causes TUT7 to bind directly to the stem-loop structure on Zc3h12a 3′-UTR, thereby promotes Zc3h12a uridylation and degradation. Zc3h12a from LPS-treated TUT7-sufficient macrophages possesses increased oligo-uridylated ends with shorter poly(A) tails, whereas oligo-uridylated Zc3h12a is significantly reduced in Tut7-/- cells after TLR4 activation. Together, our findings reveal the functional role of TUT7 in sculpting TLR4-driven responses by modulating mRNA stability of a selected set of inflammatory mediators.

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Identification and characterization of a sequence motif involved in nonsense-mediated mRNA decay.

Molecular and Cellular Biology ◽

10.1128/mcb.15.4.2231 ◽

1995 ◽

Vol 15 (4) ◽

pp. 2231-2244 ◽

Cited By ~ 67

Author(s):

S Zhang ◽

M J Ruiz-Echevarria ◽

Y Quan ◽

S W Peltz

Keyword(s):

Mrna Decay ◽

Sequence Motif ◽

Nonsense Mutations ◽

Stem Loop ◽

Cis Acting ◽

Stem Loop Structure ◽

Nonsense Codon ◽

Nonsense Mediated Mrna Decay

In both prokaryotes and eukaryotes, nonsense mutations in a gene can enhance the decay rate or reduce the abundance of the mRNA transcribed from that gene, and we call this process nonsense-mediated mRNA decay. We have been investigating the cis-acting sequences involved in this decay pathway. Previous experiments have demonstrated that, in addition to a nonsense codon, specific sequences 3' of a nonsense mutation, which have been defined as downstream elements, are required for mRNA destabilization. The results presented here identify a sequence motif (TGYYGATGYYYYY, where Y stands for either T or C) that can predict regions in genes that, when positioned 3' of a nonsense codon, promote rapid decay of its mRNA. Sequences harboring two copies of the motif from five regions in the PGK1, ADE3, and HIS4 genes were able to function as downstream elements. In addition, four copies of this motif can function as an independent downstream element. The sequences flanking the motif played a more significant role in modulating its activity when fewer copies of the sequence motif were present. Our results indicate the sequences 5' of the motif can modulate its activity by maintaining a certain distance between the sequence motif and the termination codon. We also suggest that the sequences 3' of the motif modulate the activity of the downstream element by forming RNA secondary structures. Consistent with this view, a stem-loop structure positioned 3' of the sequence motif can enhance the activity of the downstream element. This sequence motif is one of the few elements that have been identified that can predict regions in genes that can be involved in mRNA turnover. The role of these sequences in mRNA decay is discussed.

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sRNA STnc150 is involved in virulence regulation of Salmonella Typhimurium by targeting fimA mRNA

FEMS Microbiology Letters ◽

10.1093/femsle/fnab124 ◽

2021 ◽

Vol 368 (18) ◽

Author(s):

Jing Li ◽

Na Li ◽

Chengcheng Ning ◽

Yun Guo ◽

Chunhui Ji ◽

...

Keyword(s):

Salmonella Typhimurium ◽

Target Genes ◽

Reporter System ◽

Stem Loop ◽

Viral Loads ◽

Virulence Regulation ◽

Stem Loop Structure ◽

Complementary Strain

ABSTRACT Small RNAs (sRNAs) are essential virulent regulators in Salmonella typhimurium (STM). To explore the role of sRNA STnc150 in regulating STM virulence, we constructed a STnc150 deletion strain (ΔSTnc150) and its complementary strain (ΔSTnc150/C). Then, we compared their characteristics to their original parent strain experimentally, identified the target genes of STnc150 and determined the expression levels of target genes. The results showed that the ΔSTnc150 strain exhibited delayed biofilm formation, enhanced adhesion to macrophages, significantly reduced LD50, increased liver and spleen viral loads and more vital pathological damaging ability than its parent and complementary strains. Further, bioinformatics combined with the bacterial dual plasmid reporter system confirmed that the bases 72–88 of STnc150 locating at the secondary stem-loop structure of the STnc150 are complementary with the bases 1–19 in the 5′-terminal of fimA mRNA of the type 1 fimbriae subunit. Western blot analysis showed that fimA protein level was increased in STnc150 strain compared with its parent and complementary strains. Together, this study suggested that STnc150 can down-regulate STM fimA expression at the translation level, which provided insights into the regulatory mechanisms of sRNAs in virulence of STM.

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The control of inflammation via the phosphorylation and dephosphorylation of tristetraprolin: a tale of two phosphatases

Biochemical Society Transactions ◽

10.1042/bst20160166 ◽

2016 ◽

Vol 44 (5) ◽

pp. 1321-1337 ◽

Cited By ~ 39

Author(s):

Andrew R. Clark ◽

Jonathan L.E. Dean

Keyword(s):

Inflammatory Mediators ◽

Knockout Mouse ◽

Rna Binding ◽

Binding Protein ◽

Rna Binding Protein ◽

Human Orthologue ◽

And Function ◽

Detailed Picture ◽

Lines Of Evidence

Twenty years ago, the first description of a tristetraprolin (TTP) knockout mouse highlighted the fundamental role of TTP in the restraint of inflammation. Since then, work from several groups has generated a detailed picture of the expression and function of TTP. It is a sequence-specific RNA-binding protein that orchestrates the deadenylation and degradation of several mRNAs encoding inflammatory mediators. It is very extensively post-translationally modified, with more than 30 phosphorylations that are supported by at least two independent lines of evidence. The phosphorylation of two particular residues, serines 52 and 178 of mouse TTP (serines 60 and 186 of the human orthologue), has profound effects on the expression, function and localisation of TTP. Here, we discuss the control of TTP biology via its phosphorylation and dephosphorylation, with a particular focus on recent advances and on questions that remain unanswered.

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Specificity of RNA Binding by CPEB: Requirement for RNA Recognition Motifs and a Novel Zinc Finger

Molecular and Cellular Biology ◽

10.1128/mcb.18.2.685 ◽

1998 ◽

Vol 18 (2) ◽

pp. 685-693 ◽

Cited By ~ 108

Author(s):

Laura E. Hake ◽

Raul Mendez ◽

Joel D. Richter

Keyword(s):

Zinc Finger ◽

Rna Binding ◽

Rna Recognition ◽

Cytoplasmic Polyadenylation ◽

Functional Motif ◽

Rna Recognition Motifs ◽

Amino Terminal ◽

Terminal Amino ◽

Maternal Mrnas ◽

Recognition Motifs

ABSTRACT CPEB is an RNA binding protein that interacts with the maturation-type cytoplasmic polyadenylation element (CPE) (consensus UUUUUAU) to promote polyadenylation and translational activation of maternal mRNAs in Xenopus laevis. CPEB, which is conserved from mammals to invertebrates, is composed of three regions: an amino-terminal portion with no obvious functional motif, two RNA recognition motifs (RRMs), and a cysteine-histidine region that is reminiscent of a zinc finger. In this study, we investigated the physical properties of CPEB required for RNA binding. CPEB can interact with RNA as a monomer, and phosphorylation, which modifies the protein during oocyte maturation, has little effect on RNA binding. Deletion mutations of CPEB have been overexpressed inEscherichia coli and used in a series of RNA gel shift experiments. Although a full-length and a truncated CPEB that lacks 139 amino-terminal amino acids bind CPE-containing RNA avidly, proteins that have had either RRM deleted bind RNA much less efficiently. CPEB that has had the cysteine-histidine region deleted has no detectable capacity to bind RNA. Single alanine substitutions of specific cysteine or histidine residues within this region also abolish RNA binding, pointing to the importance of this highly conserved domain of the protein. Chelation of metal ions by 1,10-phenanthroline inhibits the ability of CPEB to bind RNA; however, RNA binding is restored if the reaction is supplemented with zinc. CPEB also binds other metals such as cobalt and cadmium, but these destroy RNA binding. These data indicate that the RRMs and a zinc finger region of CPEB are essential for RNA binding.

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Distinct Poly(rC) Binding Protein KH Domain Determinants for Poliovirus Translation Initiation and Viral RNA Replication

Journal of Virology ◽

10.1128/jvi.76.23.12008-12022.2002 ◽

2002 ◽

Vol 76 (23) ◽

pp. 12008-12022 ◽

Cited By ~ 100

Author(s):

Brandon L. Walter ◽

Todd B. Parsley ◽

Ellie Ehrenfeld ◽

Bert L. Semler

Keyword(s):

Translation Initiation ◽

Rna Synthesis ◽

Rna Binding ◽

Binding Protein ◽

Rna Replication ◽

Viral Rna ◽

Host Protein ◽

Loop Structure ◽

Stem Loop ◽

Stem Loop Structure

ABSTRACT The limited coding capacity of picornavirus genomic RNAs necessitates utilization of host cell factors in the completion of an infectious cycle. One host protein that plays a role in both translation initiation and viral RNA synthesis is poly(rC) binding protein 2 (PCBP2). For picornavirus RNAs containing type I internal ribosome entry site (IRES) elements, PCBP2 binds the major stem-loop structure (stem-loop IV) in the IRES and is essential for translation initiation. Additionally, the binding of PCBP2 to the 5′-terminal stem-loop structure (stem-loop I or cloverleaf) in concert with viral protein 3CD is required for initiation of RNA synthesis directed by poliovirus replication complexes. PCBP1, a highly homologous isoform of PCBP2, binds to poliovirus stem-loop I with an affinity similar to that of PCBP2; however, PCBP1 has reduced affinity for stem-loop IV. Using a dicistronic poliovirus RNA, we were able to functionally uncouple translation and RNA replication in PCBP-depleted extracts. Our results demonstrate that PCBP1 rescues RNA replication but is not able to rescue translation initiation. We have also generated mutated versions of PCBP2 containing site-directed lesions in each of the three RNA-binding domains. Specific defects in RNA binding to either stem-loop I and/or stem-loop IV suggest that these domains may have differential functions in translation and RNA replication. These predictions were confirmed in functional assays that allow separation of RNA replication activities from translation. Our data have implications for differential picornavirus template utilization during viral translation and RNA replication and suggest that specific PCBP2 domains may have distinct roles in these activities.

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miR-21 is upregulated, promoting fibrosis and blocking G2/M in irradiated rat cardiac fibroblasts

PeerJ ◽

10.7717/peerj.10502 ◽

2020 ◽

Vol 8 ◽

pp. e10502

Author(s):

Huan Guo ◽

Xinke Zhao ◽

Haixiang Su ◽

Chengxu Ma ◽

Kai Liu ◽

...

Keyword(s):

Myocardial Fibrosis ◽

Target Genes ◽

Cardiac Fibrosis ◽

Cardiac Fibroblasts ◽

Differentially Expressed Gene ◽

Stem Loop ◽

Cardiac Morbidity ◽

Increased Risk ◽

And Function

Background Radiation exposure of the thorax is associated with a greatly increased risk of cardiac morbidity and mortality even after several decades of advancement in the field. Although many studies have demonstrated the damaging influence of ionizing radiation on cardiac fibroblast (CF) structure and function, myocardial fibrosis, the molecular mechanism behind this damage is not well understood. miR-21, a small microRNA, promotes the activation of CFs, leading to cardiac fibrosis. miR-21 is overexpressed after irradiation; however, the relationship between increased miR-21 and myocardial fibrosis after irradiation is unclear. This study was conducted to investigate gene expression after radiation-induced CF damage and the role of miR-21 in this process in rats. Methods We sequenced irradiated rat CFs and performed weighted correlation network analysis (WGCNA) combined with differentially expressed gene (DEG) analysis to observe the effect on the expression profile of CF genes after radiation. Results DEG analysis showed that the degree of gene changes increased with the radiation dose. WGCNA revealed three module eigengenes (MEs) associated with 8.5-Gy-radiation—the Yellow, Brown, Blue modules. The three module eigengenes were related to apoptosis, G2/M phase, and cell death and S phase, respectively. By blocking with the cardiac fibrosis miRNA miR-21, we found that miR-21 was associated with G2/M blockade in the cell cycle and was mainly involved in regulating extracellular matrix-related genes, including Grem1, Clu, Gdf15, Ccl7, and Cxcl1. Stem-loop quantitative real-time PCR was performed to verify the expression of these genes. Five genes showed higher expression after 8.5 Gy-radiation in CFs. The target genes of miR-21 predicted online were Gdf15 and Rsad2, which showed much higher expression after treatment with antagomir-miR-21 in 8.5-Gy-irradiated CFs. Thus, miR-21 may play the role of fibrosis and G2/M blockade in regulating Grem1, Clu, Gdf15, Ccl7, Cxcl1, and Rsad2 post-irradiation.

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Expanding Role of Ubiquitin in Translational Control

International Journal of Molecular Sciences ◽

10.3390/ijms21031151 ◽

2020 ◽

Vol 21 (3) ◽

pp. 1151 ◽

Cited By ~ 3

Author(s):

Shannon E. Dougherty ◽

Austin O. Maduka ◽

Toshifumi Inada ◽

Gustavo M. Silva

Keyword(s):

Translational Control ◽

Ribosomal Proteins ◽

Ribosome Biogenesis ◽

Rna Binding ◽

Protein Quality ◽

Rna Binding Proteins ◽

Ubiquitin Chain ◽

Molecular Aspects ◽

And Function

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.

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Structure-function analysis of microRNA 3′-end trimming by Nibbler

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2018156117 ◽

2020 ◽

Vol 117 (48) ◽

pp. 30370-30379

Author(s):

Wei Xie ◽

Ivica Sowemimo ◽

Rippei Hayashi ◽

Juncheng Wang ◽

Thomas R. Burkard ◽

...

Keyword(s):

Rna Binding ◽

Function Analysis ◽

Principal Role ◽

Functional Studies ◽

Surface Patches ◽

Activity Modeling ◽

Binding Surface ◽

Catalytic Cleavage ◽

Cleavage Motif

Nibbler (Nbr) is a 3′-to-5′ exoribonuclease whose catalytic 3′-end trimming activity impacts microRNA (miRNA) and PIWI-interacting RNA (piRNA) biogenesis. Here, we report on structural and functional studies to decipher the contributions of Nbr’s N-terminal domain (NTD) and exonucleolytic domain (EXO) in miRNA 3′-end trimming. We have solved the crystal structures of the NTD core and EXO domains of Nbr, both in the apo-state. The NTD-core domain ofAedes aegyptiNbr adopts a HEAT-like repeat scaffold with basic patches constituting an RNA-binding surface exhibiting a preference for binding double-strand RNA (dsRNA) over single-strand RNA (ssRNA). Structure-guided functional assays inDrosophilaS2 cells confirmed a principal role of the NTD in exonucleolytic miRNA trimming, which depends on basic surface patches. Gain-of-function experiments revealed a potential role of the NTD in recruiting Nbr to Argonaute-bound small RNA substrates. The EXO domain ofA. aegyptiandDrosophila melanogasterNbr adopt a mixed α/β-scaffold with a deep pocket lined by a DEDDy catalytic cleavage motif. We demonstrate that Nbr’s EXO domain exhibits Mn2+-dependent ssRNA-specific 3′-to-5′ exoribonuclease activity. Modeling of a 3′ terminal Uridine into the catalytic pocket of Nbr EXO indicates that 2′-O-methylation of the 3′-U would result in a steric clash with a tryptophan side chain, suggesting that 2′-O-methylation protects small RNAs from Nbr-mediated trimming. Overall, our data establish that Nbr requires its NTD as a substrate recruitment platform to execute exonucleolytic miRNA maturation, catalyzed by the ribonuclease EXO domain.

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