Drosophila Stem-Loop Binding Protein Intracellular Localization Is Mediated by Phosphorylation and Is Required for Cell Cycle-regulated Histone mRNA Expression

Stem-loop binding protein (SLBP) is an essential component of the histone pre-mRNA processing machinery. SLBP protein expression was examined during Drosophila development by using transgenes expressing hemagglutinin (HA) epitope-tagged proteins expressed from the endogenous Slbp promoter. Full-length HA-dSLBP complemented a Slbp null mutation, demonstrating that it was fully functional. dSLBP protein accumulates throughout the cell cycle, in contrast to the observed restriction of mammalian SLBP to S phase. dSLBP is located in both nucleus and cytoplasm in replicating cells, but it becomes predominantly nuclear during G2. dSLBP is present in mitotic cells and is down-regulated in G1 when cells exit the cell cycle. We determined whether mutation at previously identified phosphorylation sites, T120 and T230, affected the ability of the protein to restore viability and histone mRNA processing to dSLBP null mutants. The T120A SLBP restored viability and histone pre-mRNA processing. However, the T230A mutant, located in a conserved TPNK sequence in the RNA binding domain, did not restore viability and histone mRNA processing in vivo, although it had full activity in histone mRNA processing in vitro. The T230A protein is concentrated in the cytoplasm, suggesting that it is defective in nuclear targeting, and accounting for its failure to function in histone pre-mRNA processing in vivo.

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Phosphorylation of Stem-Loop Binding Protein (SLBP) on Two Threonines Triggers Degradation of SLBP, the Sole Cell Cycle-Regulated Factor Required for Regulation of Histone mRNA Processing, at the End of S Phase

Molecular and Cellular Biology ◽

10.1128/mcb.23.5.1590-1601.2003 ◽

2003 ◽

Vol 23 (5) ◽

pp. 1590-1601 ◽

Cited By ~ 62

Author(s):

Lianxing Zheng ◽

Zbigniew Dominski ◽

Xiao-Cui Yang ◽

Phillip Elms ◽

Christy S. Raska ◽

...

Keyword(s):

Cell Cycle ◽

Amino Acids ◽

Posttranscriptional Regulation ◽

Binding Protein ◽

S Phase ◽

Mrna Processing ◽

Histone Mrna ◽

Stem Loop ◽

Histone Mrnas ◽

Nuclear Extracts

ABSTRACT The replication-dependent histone mRNAs, the only eukaryotic mRNAs that do not have poly(A) tails, are present only in S-phase cells. Coordinate posttranscriptional regulation of histone mRNAs is mediated by the stem-loop at the 3′ end of histone mRNAs. The protein that binds the 3′ end of histone mRNA, stem-loop binding protein (SLBP), is required for histone pre-mRNA processing and is involved in multiple aspects of histone mRNA metabolism. SLBP is also regulated during the cell cycle, accumulating as cells enter S phase and being rapidly degraded as cells exit S phase. Mutation of any residues in a TTP sequence (amino acids 60 to 62) or mutation of a consensus cyclin binding site (amino acids 99 to 104) stabilizes SLBP in G2 and mitosis. These two threonines are phosphorylated in late S phase, as determined by mass spectrometry (MS) of purified SLBP from late S-phase cells, triggering SLBP degradation. Cells that express a stable SLBP still degrade histone mRNA at the end of S phase, demonstrating that degradation of SLBP is not required for histone mRNA degradation. Nuclear extracts from G1 and G2 cells are deficient in histone pre-mRNA processing, which is restored by addition of recombinant SLBP, indicating that SLBP is the only cell cycle-regulated factor required for histone pre-mRNA processing.

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Two Xenopus Proteins That Bind the 3′ End of Histone mRNA: Implications for Translational Control of Histone Synthesis during Oogenesis

Molecular and Cellular Biology ◽

10.1128/mcb.19.1.835 ◽

1999 ◽

Vol 19 (1) ◽

pp. 835-845 ◽

Cited By ~ 52

Author(s):

Zeng-Feng Wang ◽

Thomas C. Ingledue ◽

Zbigniew Dominski ◽

Ricardo Sanchez ◽

William F. Marzluff

Keyword(s):

Translational Control ◽

Mrna Processing ◽

Loop Structure ◽

Histone Mrna ◽

Stem Loop ◽

Histone Mrnas ◽

Stem Loop Structure ◽

Histone Synthesis

ABSTRACT Translationally inactive histone mRNA is stored in frog oocytes, and translation is activated at oocyte maturation. The replication-dependent histone mRNAs are not polyadenylated and end in a conserved stem-loop structure. There are two proteins (SLBPs) which bind the 3′ end of histone mRNA in frog oocytes. SLBP1 participates in pre-mRNA processing in the nucleus. SLBP2 is oocyte specific, is present in the cytoplasm, and does not support pre-mRNA processing in vivo or in vitro. The stored histone mRNA is bound to SLBP2. As oocytes mature, SLBP2 is degraded and a larger fraction of the histone mRNA is bound to SLBP1. The mechanism of activation of translation of histone mRNAs may involve exchange of SLBPs associated with the 3′ end of histone mRNA.

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Polypyrimidine tract-binding protein blocks miRNA-124 biogenesis to enforce its neuronal-specific expression in the mouse

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1809609115 ◽

2018 ◽

Vol 115 (47) ◽

pp. E11061-E11070 ◽

Cited By ~ 6

Author(s):

Kyu-Hyeon Yeom ◽

Simon Mitchell ◽

Anthony J. Linares ◽

Sika Zheng ◽

Chia-Ho Lin ◽

...

Keyword(s):

Neuronal Differentiation ◽

Rna Binding ◽

Binding Protein ◽

Embryonic Stem ◽

Specific Expression ◽

Stem Loop ◽

Polypyrimidine Tract Binding Protein ◽

Precursor Rna

MicroRNA (miRNA)-124 is expressed in neurons, where it represses genes inhibitory for neuronal differentiation, including the RNA binding protein PTBP1. PTBP1 maintains nonneuronal splicing patterns of mRNAs that switch to neuronal isoforms upon neuronal differentiation. We find that primary (pri)-miR-124-1 is expressed in mouse embryonic stem cells where mature miR-124 is absent. PTBP1 binds to this precursor RNA upstream of the miRNA stem–loop to inhibit mature miR-124 expression in vivo and DROSHA cleavage of pri-miR-124-1 in vitro. This function for PTBP1 in repressing miR-124 biogenesis defines an additional regulatory loop in the already intricate interplay between these two molecules. Applying mathematical modeling to examine the dynamics of this regulation, we find that the pool of pri-miR-124 whose maturation is blocked by PTBP1 creates a robust and self-reinforcing transition in gene expression as PTBP1 is depleted during early neuronal differentiation. While interlocking regulatory loops are often found between miRNAs and transcriptional regulators, our results indicate that miRNA targeting of posttranscriptional regulators also reinforces developmental decisions. Notably, induction of neuronal differentiation observed upon PTBP1 knockdown likely results from direct derepression of miR-124, in addition to indirect effects previously described.

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The RNA Binding Activity of a Ribosome Biogenesis Factor, Nucleophosmin/B23, Is Modulated by Phosphorylation with a Cell Cycle-dependent Kinase and by Association with Its Subtype

Molecular Biology of the Cell ◽

10.1091/mbc.02-03-0036 ◽

2002 ◽

Vol 13 (6) ◽

pp. 2016-2030 ◽

Cited By ~ 120

Author(s):

Mitsuru Okuwaki ◽

Masafumi Tsujimoto ◽

Kyosuke Nagata

Keyword(s):

Cell Cycle ◽

Ribosome Biogenesis ◽

Cellular Localization ◽

Rna Binding ◽

Intracellular Localization ◽

Binding Activity ◽

Cyclin B ◽

Cycle Dependent

Nucleophosmin/B23 is a nucleolar phosphoprotein. It has been shown that B23 binds to nucleic acids, digests RNA, and is localized in nucleolar granular components from which preribosomal particles are transported to cytoplasm. The intracellular localization of B23 is significantly changed during the cell cycle. Here, we have examined the cellular localization of B23 proteins and the effect of mitotic phosphorylation of B23.1 on its RNA binding activity. Two splicing variants of B23 proteins, termed B23.1 and B23.2, were complexed both in vivo and in vitro. The RNA binding activity of B23.1 was impaired by hetero-oligomer formation with B23.2. Both subtypes of B23 proteins were phosphorylated during mitosis by cyclin B/cdc2. The RNA binding activity of B23.1 was repressed through cyclin B/cdc2-mediated phosphorylation at specific sites in B23. Thus, the RNA binding activity of B23.1 is stringently modulated by its phosphorylation and subtype association. Interphase B23.1 was mainly localized in nucleoli, whereas B23.2 and mitotic B23.1, those of which were incapable of binding to RNA, were dispersed throughout the nucleoplasm and cytoplasm, respectively. These results suggest that nucleolar localization of B23.1 is mediated by its ability to associate with RNA.

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Phosphorylation of Threonine 61 by Cyclin A/Cdk1 Triggers Degradation of Stem-Loop Binding Protein at the End of S Phase

Molecular and Cellular Biology ◽

10.1128/mcb.01416-07 ◽

2008 ◽

Vol 28 (14) ◽

pp. 4469-4479 ◽

Cited By ~ 44

Author(s):

M. Murat Koseoglu ◽

Lee M. Graves ◽

William F. Marzluff

Keyword(s):

Cell Cycle ◽

Fusion Proteins ◽

Regulatory Mechanism ◽

Binding Protein ◽

Cyclin A ◽

S Phase ◽

Mrna Levels ◽

Histone Mrna ◽

Stem Loop

ABSTRACT Histone mRNA levels are cell cycle regulated, and a major regulatory mechanism is restriction of stem-loop binding protein (SLBP) to S phase. Degradation of SLBP at the end of S phase results in cessation of histone mRNA biosynthesis, preventing accumulation of histone mRNA until SLBP is synthesized just before entry into the next S phase. Degradation of SLBP requires an SFTTP (58 to 62) and KRKL (95 to 98) sequence, which is a putative cyclin binding site. A fusion protein with the 58-amino-acid sequence of SLBP (amino acids 51 to 108) fused to glutathione S-transferase (GST) is sufficient to mimic SLBP degradation at late S phase. Using GST-SLBP fusion proteins as a substrate, we show that cyclin A/Cdk1 phosphorylates Thr61. Furthermore, knockdown of Cdk1 by RNA interference stabilizes SLBP at the end of S phase. Phosphorylation of Thr61 is necessary for subsequent phosphorylation of Thr60 by CK2 in vitro. Inhibitors of CK2 also prevent degradation of SLBP at the end of S phase. Thus, phosphorylation of Thr61 by cyclin A/Cdk1 primes phosphorylation of Thr60 by CK2 and is responsible for initiating SLBP degradation. We conclude that the increase in cyclin A/Cdk1 activity at the end of S phase triggers degradation of SLBP at S/G2.

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Stem-Loop Binding Protein, the Protein That Binds the 3′ End of Histone mRNA, Is Cell Cycle Regulated by Both Translational and Posttranslational Mechanisms

Molecular and Cellular Biology ◽

10.1128/mcb.20.12.4188-4198.2000 ◽

2000 ◽

Vol 20 (12) ◽

pp. 4188-4198 ◽

Cited By ~ 139

Author(s):

Michael L. Whitfield ◽

Lian-Xing Zheng ◽

Amy Baldwin ◽

Tomohiko Ohta ◽

Myra M. Hurt ◽

...

Keyword(s):

Cell Cycle ◽

Cell Cycle Regulation ◽

Binding Protein ◽

S Phase ◽

Mrna Processing ◽

Mrna Levels ◽

Histone Mrna ◽

Stem Loop ◽

Histone Mrnas ◽

Cycle Regulation

ABSTRACT The expression of the replication-dependent histone mRNAs is tightly regulated during the cell cycle. As cells progress from G1 to S phase, histone mRNA levels increase 35-fold, and they decrease again during G2 phase. Replication-dependent histone mRNAs are the only metazoan mRNAs that lack polyadenylated tails, ending instead in a conserved stem-loop. Much of the cell cycle regulation is posttranscriptional and is mediated by the 3′ stem-loop. A 31-kDa stem-loop binding protein (SLBP) binds the 3′ end of histone mRNA. The SLBP is necessary for pre-mRNA processing and accompanies the histone mRNA to the cytoplasm, where it is a component of the histone messenger RNP. We used synchronous CHO cells selected by mitotic shakeoff and HeLa cells synchronized at the G1/S or the M/G1 boundary to study the regulation of SLBP during the cell cycle. In each system the amount of SLBP is regulated during the cell cycle, increasing 10- to 20-fold in the late G1 and then decreasing in the S/G2border. SLBP mRNA levels are constant during the cell cycle. SLBP is regulated at the level of translation as cells progress from G1 to S phase, and the protein is rapidly degraded as they progress into G2. Regulation of SLBP may account for the posttranscriptional component of the cell cycle regulation of histone mRNA.

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Nuclear Import of the Stem–Loop Binding Protein and Localization during the Cell Cycle

Molecular Biology of the Cell ◽

10.1091/mbc.e04-11-1023 ◽

2005 ◽

Vol 16 (6) ◽

pp. 2960-2971 ◽

Cited By ~ 24

Author(s):

Judith A. Erkmann ◽

Eric J. Wagner ◽

Jian Dong ◽

Yanping Zhang ◽

Ulrike Kutay ◽

...

Keyword(s):

Cell Cycle ◽

Nuclear Localization ◽

Nuclear Import ◽

Binding Protein ◽

Histone Mrna ◽

Stem Loop ◽

Histone Mrnas ◽

Import Receptors ◽

Mrna Binding

A key factor involved in the processing of histone pre-mRNAs in the nucleus and translation of mature histone mRNAs in the cytoplasm is the stem–loop binding protein (SLBP). In this work, we have investigated SLBP nuclear transport and subcellular localization during the cell cycle. SLBP is predominantly nuclear under steady-state conditions and localizes to the cytoplasm during S phase when histone mRNAs accumulate. Consistently, SLBP mutants that are defective in histone mRNA binding remain nuclear. As assayed in heterokaryons, export of SLBP from the nucleus is dependent on histone mRNA binding, demonstrating that SLBP on its own does not possess any nuclear export signals. We find that SLBP interacts with the import receptors Impα/Impβ and Transportin-SR2. Moreover, complexes formed between SLBP and the two import receptors are disrupted by RanGTP. We have further shown that SLBP is imported by both receptors in vitro. Three sequences in SLBP required for Impα/Impβ binding were identified. Simultaneous mutation of all three sequences was necessary to abolish SLBP nuclear localization in vivo. In contrast, we were unable to identify an in vivo role for Transportin-SR2 in SLBP nuclear localization. Thus, only the Impα/Impβ pathway contributes to SLBP nuclear import in HeLa cells.

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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.

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EWSR1-induced circNEIL3 promotes glioma progression and exosome-mediated macrophage immunosuppressive polarization via stabilizing IGF2BP3

Molecular Cancer ◽

10.1186/s12943-021-01485-6 ◽

2022 ◽

Vol 21 (1) ◽

Author(s):

Ziwen Pan ◽

Rongrong Zhao ◽

Boyan Li ◽

Yanhua Qi ◽

Wei Qiu ◽

...

Keyword(s):

Rna Binding ◽

Binding Protein ◽

Expression Profiles ◽

Tumour Microenvironment ◽

Malignant Progression ◽

Luciferase Reporter ◽

Sequencing Analysis ◽

Glioma Progression

Abstract Background Gliomas are the most common malignant primary brain tumours with a highly immunosuppressive tumour microenvironment (TME) and poor prognosis. Circular RNAs (circRNA), a newly found type of endogenous noncoding RNA, characterized by high stability, abundance, conservation, have been shown to play an important role in the pathophysiological processes and TME remodelling of various tumours. Methods CircRNA sequencing analysis was performed to explore circRNA expression profiles in normal and glioma tissues. The biological function of a novel circRNA, namely, circNEIL3, in glioma development was confirmed both in vitro and in vivo. Mechanistically, RNA pull-down, mass spectrum, RNA immunoprecipitation (RIP), luciferase reporter, and co-immunoprecipitation assays were conducted. Results We identified circNEIL3, which could be cyclized by EWS RNA-binding protein 1(EWSR1), to be upregulated in glioma tissues and to correlate positively with glioma malignant progression. Functionally, we confirmed that circNEIL3 promotes tumorigenesis and carcinogenic progression of glioma in vitro and in vivo. Mechanistically, circNEIL3 stabilizes IGF2BP3 (insulin-like growth factor 2 mRNA binding protein 3) protein, a known oncogenic protein, by preventing HECTD4-mediated ubiquitination. Moreover, circNEIL3 overexpression glioma cells drives macrophage infiltration into the tumour microenvironment (TME). Finally, circNEIL3 is packaged into exosomes by hnRNPA2B1 and transmitted to infiltrated tumour associated macrophages (TAMs), enabling them to acquire immunosuppressive properties by stabilizing IGF2BP3 and in turn promoting glioma progression. Conclusions This work reveals that circNEIL3 plays a nonnegligible multifaceted role in promoting gliomagenesis, malignant progression and macrophage tumour-promoting phenotypes polarization, highlighting that circNEIL3 is a potential prognostic biomarker and therapeutic target in glioma.

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