Mammalian Peptidylglycine α-Amidating Monooxygenase mRNA Expression Can Be Modulated by the La Autoantigen

ABSTRACT Peptidylglycine α-amidating monooxygenase (PAM; EC 1.14.17.3) catalyzes the COOH-terminal α-amidation of peptidylglycine substrates, yielding amidated products. We have previously reported a putative regulatory RNA binding protein (PAM mRNA-BP) that binds specifically to the 3′ untranslated region (UTR) of PAM-mRNA. Here, the PAM mRNA-BP was isolated and revealed to be La protein using affinity purification onto a 3′ UTR PAM RNA, followed by tandem mass spectrometry identification. We determined that the core binding sequence is approximately 15-nucleotides (nt) long and is located 471 nt downstream of the stop codon. Moreover, we identified the La autoantigen as a protein that specifically binds the 3′ UTR of PAM mRNA in vivo and in vitro. Furthermore, La protein overexpression caused a nuclear retention of PAM mRNAs and resulted in the down-regulation of endogenous PAM activity. Most interestingly, the nuclear retention of PAM mRNA is lost upon expressing the La proteins that lack a conserved nuclear retention element, suggesting a direct association between PAM mRNA and La protein in vivo. Reporter assays using a chimeric mRNA that combined luciferase and the 3′ UTR of PAM mRNA demonstrated a decrease of the reporter activity due to an increase in the nuclear localization of reporter mRNAs, while the deletion of the 15-nt La binding site led to their clear-cut cytoplasmic relocalization. The results suggest an important role for the La protein in the modulation of PAM expression, possibly by mechanisms that involve a nuclear retention and perhaps a processing of pre-PAM mRNA molecules.

Download Full-text

Hypogonadism associated withCyp19a1 (Aromatase) post-transcriptional upregulation inCelf1-KO mice

Molecular and Cellular Biology ◽

10.1128/mcb.00074-15 ◽

2015 ◽

pp. MCB.00074-15 ◽

Cited By ~ 2

Author(s):

Gaella Boulanger ◽

Marie Cibois ◽

Justine Viet ◽

Alexis Fostier ◽

Stéphane Deschamps ◽

...

Keyword(s):

Rna Binding ◽

Hypogonadotropic Hypogonadism ◽

Type I ◽

Male Gametes ◽

Low Concentrations ◽

Reporter Assays ◽

High Concentrations ◽

In Vitro Interaction

CELF1 is a multifunctional RNA-binding protein that controls several aspects of RNA fate. The targeted disruption of theCelf1gene in mice causes male infertility due to impaired spermiogenesis, the post-meiotic differentiation of male gametes. Here, we investigated the molecular reasons that underlie this testicular phenotype. By measuring sex hormone levels, we detected low concentrations of testosterone inCelf1-null mice. We investigated the effect ofCelf1disruption on the expression levels of steroidogenic enzyme genes, and we observed thatCyp19a1was upregulated.Cyp19a1encodes aromatase, which transforms testosterone into estradiol. Administration of testosterone or the aromatase inhibitor Letrozole partly rescued the spermiogenesis defects, indicating that a lack of testosterone associated with excessive aromatase contributes to the testicular phenotype. In vivo and in vitro interaction assays demonstrated that CELF1 binds toCyp19a1mRNA, and reporter assays supported the conclusion that CELF1 directly repressesCyp19a1translation. We conclude that CELF1 downregulatesCyp19a1/Aromatasepost-transcriptionally to achieve high concentrations of testosterone compatible with spermiogenesis completion. We discuss the implications of these findings with respect to reproductive defects in men, including patients suffering from isolated hypogonadotropic hypogonadism and myotonic dystrophy type I.

Download Full-text

SYNCRIP, a Member of the Heterogeneous Nuclear Ribonucleoprotein Family, Is Involved in Mouse Hepatitis Virus RNA Synthesis

Journal of Virology ◽

10.1128/jvi.78.23.13153-13162.2004 ◽

2004 ◽

Vol 78 (23) ◽

pp. 13153-13162 ◽

Cited By ~ 37

Author(s):

Keum S. Choi ◽

Akihiro Mizutani ◽

Michael M. C. Lai

Keyword(s):

Mammalian Cells ◽

Rna Synthesis ◽

Rna Binding ◽

Hepatitis Virus ◽

Affinity Purification ◽

Mouse Hepatitis Virus ◽

Viral Rna ◽

Negative Strand

ABSTRACT Several cellular proteins, including several heterogeneous nuclear ribonucleoproteins (hnRNPs), have been shown to function as regulatory factors for mouse hepatitis virus (MHV) RNA synthesis as a result of their binding to the 5′ and 3′ untranslated regions (UTRs) of the viral RNA. Here, we identified another cellular protein, p70, which has been shown by UV cross-linking to bind both the positive- and negative-strand UTRs of MHV RNA specifically. We purified p70 with a a one-step RNA affinity purification procedure with the biotin-labeled 5′-UTR. Matrix-assisted laser desorption ionization (MALDI)-mass spectrometry identified it as synaptotagmin-binding cytoplasmic RNA-interacting protein (SYNCRIP). SYNCRIP is a member of the hnRNP family and localizes largely in the cytoplasm. The p70 was cross-linked to the MHV positive- or negative-strand UTR in vitro and in vivo. The bacterially expressed SYNCRIP was also able to bind to the 5′-UTR of both strands. The SYNCRIP-binding site was mapped to the leader sequence of the 5′-UTR, requiring the UCUAA repeat sequence. To investigate the functional significance of SYNCRIP in MHV replication, we expressed a full-length or a C-terminally truncated form of SYNCRIP in mammalian cells expressing the MHV receptor. The overexpression of either form of SYNCRIP inhibited syncytium formation induced by MHV infection. Furthermore, downregulation of the endogenous SYNCRIP with a specific short interfering RNA delayed MHV RNA synthesis; in contrast, overexpression or downregulation of SYNCRIP did not affect MHV translation. These results suggest that SYNCRIP may be directly involved in MHV RNA replication as a positive regulator. This study identified an additional cellular hnRNP as an MHV RNA-binding protein potentially involved in viral RNA synthesis.

Download Full-text

Cell type specific profiling of alternative translation identifies novel protein isoforms in the mouse brain

10.1101/324236 ◽

2018 ◽

Author(s):

Darshan Sapkota ◽

Allison M. Lake ◽

Wei Yang ◽

Chengran Yang ◽

Hendrik Wesseling ◽

...

Keyword(s):

Mouse Brain ◽

Stop Codon ◽

Affinity Purification ◽

Protein Isoforms ◽

Cell Type ◽

Alternative Translation ◽

Cell Type Specific ◽

Ribosome Footprinting

AbstractTranslation canonically begins at a single AUG and terminates at the stop codon, generating one protein species per transcript. However, some transcripts may use alternative initiation sites or sustain translation past their stop codon, generating multiple protein isoforms. Through other mechanisms such as alternative splicing, both neurons and glia exhibit remarkable transcriptional diversity, and these other forms of post-transcriptional regulation are impacted by neural activity and disease. Here, using ribosome footprinting, we demonstrate that alternative translation is likewise abundant in the central nervous system and modulated by stimulation and disease. First, in neuron/glia mixed cultures we identify hundreds of transcripts with alternative initiation sites and confirm the protein isoforms corresponding to a subset of these sites by mass spectrometry. Many of them modulate their alternative initiation in response to KCl stimulation, indicating activity-dependent regulation of this phenomenon. Next, we detect several transcripts undergoing stop codon readthrough thus generating novel C-terminally-extended protein isoforms in vitro. Further, by coupling Translating Ribosome Affinity Purification to ribosome footprinting to enable cell-type specific analysis in vivo, we find that several of both neuronal and astrocytic transcripts undergo readthrough in the mouse brain. Functional analyses of one of these transcripts, Aqp4, reveals readthrough confers perivascular localization, indicating readthrough can be a conserved mechanism to modulate protein function. Finally, we show that AQP4 readthrough is disrupted in multiple gliotic disease models. Our study demonstrates the extensive and regulated use of alternative translational events in the brain and indicates that some of these events alter key protein properties.

Download Full-text

Sense-overlapping lncRNA as a decoy of translational repressor protein for dimorphic gene expression

PLoS Genetics ◽

10.1371/journal.pgen.1009683 ◽

2021 ◽

Vol 17 (7) ◽

pp. e1009683

Author(s):

Christelle Alexa Garcia Perez ◽

Shungo Adachi ◽

Quang Dang Nong ◽

Nikko Adhitama ◽

Tomoaki Matsuura ◽

...

Keyword(s):

Rna Binding ◽

Binding Protein ◽

Affinity Purification ◽

Translation Efficiency ◽

Gene Expressions ◽

Translational Repressor ◽

Sense Orientation ◽

Dimorphic Gene Expression

Long noncoding RNAs (lncRNAs) are vastly transcribed and extensively studied but lncRNAs overlapping with the sense orientation of mRNA have been poorly studied. We analyzed the lncRNA DAPALR overlapping with the 5´ UTR of the Doublesex1 (Dsx1), the male determining gene in Daphnia magna. By affinity purification, we identified an RNA binding protein, Shep as a DAPALR binding protein. Shep also binds to Dsx1 5´ UTR by recognizing the overlapping sequence and suppresses translation of the mRNA. In vitro and in vivo analyses indicated that DAPALR increased Dsx1 translation efficiency by sequestration of Shep. This regulation was impaired when the Shep binding site in DAPALR was deleted. These results suggest that Shep suppresses the unintentional translation of Dsx1 by setting a threshold; and when the sense lncRNA DAPALR is expressed, DAPALR cancels the suppression caused by Shep. This mechanism may be important to show dimorphic gene expressions such as sex determination and it may account for the binary expression in various developmental processes.

Download Full-text

Mpe1, a Zinc Knuckle Protein, Is an Essential Component of Yeast Cleavage and Polyadenylation Factor Required for the Cleavage and Polyadenylation of mRNA

Molecular and Cellular Biology ◽

10.1128/mcb.21.24.8346-8356.2001 ◽

2001 ◽

Vol 21 (24) ◽

pp. 8346-8356 ◽

Cited By ~ 27

Author(s):

Le Thuy Anh Vo ◽

Michèle Minet ◽

Jean-Marie Schmitter ◽

François Lacroute ◽

Françoise Wyers

Keyword(s):

Rna Binding ◽

Essential Gene ◽

Affinity Purification ◽

Protein A ◽

Gene Encoding ◽

Cleavage And Polyadenylation ◽

The Stability ◽

Polyadenylation Factor

ABSTRACT In Saccharomyces cerevisiae, in vitro mRNA cleavage and polyadenylation require the poly(A) binding protein, Pab1p, and two multiprotein complexes: CFI (cleavage factor I) and CPF (cleavage and polyadenylation factor). We characterized a novel essential gene,MPE1 (YKL059c), which interacts genetically with the PCF11 gene encoding a subunit of CFI. Mpe1p is an evolutionarily conserved protein, a homolog of which is encoded by the human genome. The protein sequence contains a putative RNA-binding zinc knuckle motif. MPE1 is implicated in the choice ofACT1 mRNA polyadenylation site in vivo. Extracts from a conditional mutant, mpe1-1, or from a wild-type extract immunoneutralized for Mpe1p are defective in 3′-end processing. We used the tandem affinity purification (TAP) method on strains TAP-tagged for Mpe1p or Pfs2p to show that Mpe1p, like Pfs2p, is an integral subunit of CPF. Nevertheless a stable CPF, devoid of Mpe1p, was purified from the mpe1-1 mutant strain, showing that Mpe1p is not directly involved in the stability of this complex. Consistently, Mpe1p is also not necessary for the processive polyadenylation, nonspecific for the genuine pre-mRNA 3′ end, displayed by the CPF alone. However, a reconstituted assay with purified CFI, CPF, and the recombinant Pab1p showed that Mpe1p is strictly required for the specific cleavage and polyadenylation of pre-mRNA. These results show that Mpe1p plays a crucial role in 3′ end formation probably by promoting the specific link between the CFI/CPF complex and pre-mRNA.

Download Full-text

Conservation of a Masked Nuclear Export Activity of La Proteins and Its Effects on tRNA Maturation

Molecular and Cellular Biology ◽

10.1128/mcb.00026-07 ◽

2007 ◽

Vol 27 (9) ◽

pp. 3303-3312 ◽

Cited By ~ 25

Author(s):

Mark A. Bayfield ◽

Trish E. Kaiser ◽

Robert V. Intine ◽

Richard J. Maraia

Keyword(s):

Fission Yeast ◽

Nuclear Export ◽

Rna Binding ◽

Nuclear Accumulation ◽

Rna Recognition Motif ◽

Nuclear Retention ◽

Trna Processing ◽

Export Activity ◽

La Protein

ABSTRACT La is an RNA-processing-associated phosphoprotein so highly conserved that the human La protein (hLa) can replace the tRNA-processing function of the fission yeast La protein (Sla1p) in vivo. La proteins contain multiple trafficking elements that support interactions with RNAs in different subcellular locations. Prior data indicate that deletion of a nuclear retention element (NRE) causes nuclear export of La and dysfunctional processing of associated pre-tRNAs that are spliced but 5′ and 3′ unprocessed, with an accompanying decrease in tRNA-mediated suppression, in fission yeast. To further pursue these observations, we first identified conserved residues in the NREs of hLa and Sla1p that when substituted mimic the NRE deletion phenotype. NRE-defective La proteins then deleted of other motifs indicated that RNA recognition motif 1 (RRM1) is required for nuclear export. Mutations of conserved RRM1 residues restored nuclear accumulation of NRE-defective La proteins. Some RRM1 mutations restored nuclear accumulation, prevented disordered pre-tRNA processing, and restored suppression, indicating that the tRNA-related activity of RRM1 and its nuclear export activity could be functionally separated. When mapped onto an hLa structure, the export-sensitive residues comprised surfaces distinct from the RNA-binding surface of RRM1. The data indicate that the NRE has been conserved to mask or functionally override an equally conserved nuclear export activity of RRM1. The data suggest that conserved elements mediate nuclear retention, nuclear export, and RNA-binding activities of the multifunctional La protein and that their interrelationship contributes to the ability of La to engage its different classes of RNA ligands in different cellular locations.

Download Full-text

USP9X-mediated KDM4C deubiquitination promotes lung cancer radioresistance by epigenetically inducing TGF-β2 transcription

Cell Death and Differentiation ◽

10.1038/s41418-021-00740-z ◽

2021 ◽

Author(s):

Xiaohua Jie ◽

William Pat Fong ◽

Rui Zhou ◽

Ye Zhao ◽

Yingchao Zhao ◽

...

Keyword(s):

Lung Cancer ◽

Affinity Purification ◽

Smad Signaling ◽

Lung Cancer Patients ◽

Lysine Specific Demethylase ◽

Underlying Mechanisms ◽

H3k9me3 Level ◽

Critical Binding

AbstractRadioresistance is regarded as the main barrier to effective radiotherapy in lung cancer. However, the underlying mechanisms of radioresistance remain elusive. Here, we show that lysine-specific demethylase 4C (KDM4C) is overexpressed and correlated with poor prognosis in lung cancer patients. We provide evidence that genetical or pharmacological inhibition of KDM4C impairs tumorigenesis and radioresistance in lung cancer in vitro and in vivo. Moreover, we uncover that KDM4C upregulates TGF-β2 expression by directly reducing H3K9me3 level at the TGF-β2 promoter and then activates Smad/ATM/Chk2 signaling to confer radioresistance in lung cancer. Using tandem affinity purification technology, we further identify deubiquitinase USP9X as a critical binding partner that deubiquitinates and stabilizes KDM4C. More importantly, depletion of USP9X impairs TGF-β2/Smad signaling and radioresistance by destabilizing KDM4C in lung cancer cells. Thus, our findings demonstrate that USP9X-mediated KDM4C deubiquitination activates TGF-β2/Smad signaling to promote radioresistance, suggesting that targeting KDM4C may be a promising radiosensitization strategy in the treatment of lung cancer.

Download Full-text

The methyltransferase SETD2 couples transcription and splicing by engaging mRNA processing factors through its SHI domain

Nature Communications ◽

10.1038/s41467-021-21663-w ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Saikat Bhattacharya ◽

Michaella J. Levy ◽

Ning Zhang ◽

Hua Li ◽

Laurence Florens ◽

...

Keyword(s):

Rna Splicing ◽

Rna Binding ◽

Mrna Processing ◽

Key Factors ◽

Pol Ii ◽

Mrna Transcripts ◽

Hnrnp Protein ◽

Processing Factors

AbstractHeterogeneous ribonucleoproteins (hnRNPs) are RNA binding molecules that are involved in key processes such as RNA splicing and transcription. One such hnRNP protein, hnRNP L, regulates alternative splicing (AS) by binding to pre-mRNA transcripts. However, it is unclear what factors contribute to hnRNP L-regulated AS events. Using proteomic approaches, we identified several key factors that co-purify with hnRNP L. We demonstrate that one such factor, the histone methyltransferase SETD2, specifically interacts with hnRNP L in vitro and in vivo. This interaction occurs through a previously uncharacterized domain in SETD2, the SETD2-hnRNP Interaction (SHI) domain, the deletion of which, leads to a reduced H3K36me3 deposition. Functionally, SETD2 regulates a subset of hnRNP L-targeted AS events. Our findings demonstrate that SETD2, by interacting with Pol II as well as hnRNP L, can mediate the crosstalk between the transcription and the splicing machinery.

Download Full-text

Oncogenic lncRNA ZNF561-AS1 is essential for colorectal cancer proliferation and survival through regulation of miR-26a-3p/miR-128-5p-SRSF6 axis

Journal of Experimental & Clinical Cancer Research ◽

10.1186/s13046-021-01882-1 ◽

2021 ◽

Vol 40 (1) ◽

Author(s):

Zizhen Si ◽

Lei Yu ◽

Haoyu Jing ◽

Lun Wu ◽

Xidi Wang

Keyword(s):

Colorectal Cancer ◽

Rna Binding ◽

Xenograft Model ◽

Luciferase Reporter ◽

Cancer Proliferation ◽

Mouse Xenograft ◽

Mouse Xenograft Model

Abstract Background Long non-coding RNAs (lncRNA) are reported to influence colorectal cancer (CRC) progression. Currently, the functions of the lncRNA ZNF561 antisense RNA 1 (ZNF561-AS1) in CRC are unknown. Methods ZNF561-AS1 and SRSF6 expression in CRC patient samples and CRC cell lines was evaluated through TCGA database analysis, western blot along with real-time PCR. SRSF6 expression in CRC cells was also examined upon ZNF561-AS1 depletion or overexpression. Interaction between miR-26a-3p, miR-128-5p, ZNF561-AS1, and SRSF6 was examined by dual luciferase reporter assay, as well as RNA binding protein immunoprecipitation (RIP) assay. Small interfering RNA (siRNA) mediated knockdown experiments were performed to assess the role of ZNF561-AS1 and SRSF6 in the proliferative actives and apoptosis rate of CRC cells. A mouse xenograft model was employed to assess tumor growth upon ZNF561-AS1 knockdown and SRSF6 rescue. Results We find that ZNF561-AS1 and SRSF6 were upregulated in CRC patient tissues. ZNF561-AS1 expression was reduced in tissues from treated CRC patients but upregulated in CRC tissues from relapsed patients. SRSF6 expression was suppressed and enhanced by ZNF561-AS1 depletion and overexpression, respectively. Mechanistically, ZNF561-AS1 regulated SRSF6 expression by sponging miR-26a-3p and miR-128-5p. ZNF561-AS1-miR-26a-3p/miR-128-5p-SRSF6 axis was required for CRC proliferation and survival. ZNF561-AS1 knockdown suppressed CRC cell proliferation and triggered apoptosis. ZNF561-AS1 depletion suppressed the growth of tumors in a model of a nude mouse xenograft. Similar observations were made upon SRSF6 depletion. SRSF6 overexpression reversed the inhibitory activities of ZNF561-AS1 in vivo, as well as in vitro. Conclusion In summary, we find that ZNF561-AS1 promotes CRC progression via the miR-26a-3p/miR-128-5p-SRSF6 axis. This study reveals new perspectives into the role of ZNF561-AS1 in CRC.

Download Full-text

Modulation of Viral Programmed Ribosomal Frameshifting and Stop Codon Readthrough by the Host Restriction Factor Shiftless

Viruses ◽

10.3390/v13071230 ◽

2021 ◽

Vol 13 (7) ◽

pp. 1230

Author(s):

Sawsan Napthine ◽

Chris H. Hill ◽

Holly C. M. Nugent ◽

Ian Brierley

Keyword(s):

Rna Binding ◽

Mutational Analysis ◽

Stop Codon ◽

Leukemia Virus ◽

Size Exclusion ◽

Human Immunodeficiency Virus Replication ◽

Mobility Shift ◽

Ribosomal Frameshifting ◽

Stop Codon Readthrough

The product of the interferon-stimulated gene C19orf66, Shiftless (SHFL), restricts human immunodeficiency virus replication through downregulation of the efficiency of the viral gag/pol frameshifting signal. In this study, we demonstrate that bacterially expressed, purified SHFL can decrease the efficiency of programmed ribosomal frameshifting in vitro at a variety of sites, including the RNA pseudoknot-dependent signals of the coronaviruses IBV, SARS-CoV and SARS-CoV-2, and the protein-dependent stimulators of the cardioviruses EMCV and TMEV. SHFL also reduced the efficiency of stop-codon readthrough at the murine leukemia virus gag/pol signal. Using size-exclusion chromatography, we confirm the binding of the purified protein to mammalian ribosomes in vitro. Finally, through electrophoretic mobility shift assays and mutational analysis, we show that expressed SHFL has strong RNA binding activity that is necessary for full activity in the inhibition of frameshifting, but shows no clear specificity for stimulatory RNA structures.

Download Full-text