DOCSLIB.ORG

Anti-SLBP Code No

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Anti-SLBP Code No For Research Use Only. RN045P Not for use in diagnostic procedures. Page 1 of 4 RIP-Certified Antibody Anti-SLBP Code No. Quantity Concentration Form RN045P 200 L 1 mg/mL Affinity Purified BACKGROUND: The stem-loop binding protein APPLICATIONS: (SLBP) is an RNA binding protein known as a cell-cycle RNP Immunoprecipitation; 15 g/500 L of cell extract regulator. SLBP binds to the stem-loop structure of from 2.1 x 107 cells replication-dependent histone mRNA that has neither Western blotting; 1:1,000 for chemiluminescence detection introns nor poly(A) tail. SLBP is required for histone system pre-mRNA processing, histone mRNA export, translation, Immunoprecipitation; 1.5 g/500 L of cell extract from and degradation. SLBP is accumulated just before entry of 2.1 x 106 cells S phase and degraded at S/G2 transition. Cyclin A/Cdk1 Immunohistochemistry; Not tested modulates degradation of SLBP at the end of S phase, Immunocytochemistry; Not tested which leads to an inhibition of histone mRNA biosynthesis. Flow cytometry; Not tested RIP-CERTIFIED ANTIBODY: Detailed procedures are provided in the following Posttranscriptional regulation of gene expression is a PROTOCOLS. ribonucleoprotein-driven process, which involves RNA binding proteins (RBPs) and non-coding RNAs that affect REFERENCES: splicing, nuclear export, subcellular localization, mRNA 1) Sullivan, K. D., et al., RNA 15, 459-472 (2009) decay and translation. The RNP Immunoprecipitation-Chip 2) Koseoglu, M. M., et al., Mol. Cell Biol. 28, 4469-4479 (2008) (RIP-Chip), RIP-Seq and RIP-RTPCR allow the 3) Dominski, Z., et al., Genes Dev. 16, 58-71 (2002) identification of multiple RNA targets of RBPs globally 4) Wang, Z. F., et al., Genes Dev. 10, 3028-3040 (1996) and within the context of a cell extract. Antibodies specific to the RNA binding protein of interest are used to SPECIES CROSS REACTIVITY: co-immunoprecipitate the RNA binding protein and the associated subset of mRNAs. The mRNA content is Species Human Mouse Rat Hamster interrogated using standard microarray or sequencing 293T, HeLa, technology. RIP-Certified Antibody is validated for use in NIH/3T3, Cells K562, Jurkat, Rat1 CHO WR19L RNP Immunoprecipitation (RIP) in conjunction with the MCF7 RIP-Assay Kit distributed from MBL. Its ability to Reactivity on + + + + immunoprecipitate mRNAs and RBPs complex was WB confirmed by quantitative and qualitative analysis on NanoDrop, Bioanalyzer and RT-PCR or microarray. LICENSING OPPORTUNITY: The RIP-Assay uses patented technology (US patent No. 6,635,422, US patent SOURCE: This antibody was purified from rabbit serum No. 7,504,210) of Ribonomics, Inc. MBL manufactures and by affinity column chromatography. The rabbit was distributes this product under license from Ribonomics, Inc. immunized with KLH conjugated synthetic peptide, Researchers may use this product for their own research. corresponding to C-terminus of human SLBP. Researchers are not allowed to use this product or RIP-Assay technology for commercial purpose without a FORMULATION: 200 L volume of PBS containing license. For commercial use, please contact us for licensing 50% glycerol, pH 7.2. No preservative is contained. opportunities at [email protected] STORAGE: This antibody solution is stable for one year o from the date of purchase when stored at -20 C. REACTIVITY: This antibody reacts with human SLBP on Western blotting, Immunoprecipitation and RNP Immunoprecipitation. Other species cross reactivity is confirmed by Western blotting. INTENDED USE: For Research Use Only. Not for use in diagnostic procedures. MEDICAL & BIOLOGICAL LABORATORIES CO., LTD. URL https://ruo.mbl.co.jp/je/rip-assay/ e-mail [email protected], TEL 052-238-1904 RN045P Page 2 of 4 Normal Rabbit IgG Anti-SLBP resuspended in nuclease-free PBS with Normal Rabbit IgG (RIP-Assay Kit) or anti-SLBP antibody at the concentration as suggested in the APPLICATIONS, and then add 1 mL of Wash buffer (+) into each tube. Incubate with gently agitation for 1 hour at 4oC. 6) Wash the beads once with ice-cold Lysis Buffer (+) RNA Intensity RNA (centrifuge the tube at 2,000 x g for 1 minute). Carefully Nucleotide length discard the supernatant using a pipettor without disturbing the beads. G 7) Add 500 L of cell lysate (precleared sample of step 4), Ig o then incubate with gentle agitation for 3 hours at 4 C. it b b 8) Wash the beads 4 times with Wash Buffer (+) (centrifuge a P R A B N the tube at 2,000 x g for 1 minute). r l L e a R -S l d m i a 9) Add 400 L of Master mix solution (Solution I: Solution d r t t a o n o L N a T II = 10 L: 390 L). Vortex thoroughly, then spin-down. 10) Add 250 L of Solution III. Vortex thoroughly. 28 S 11) Centrifuge the tube at 2,000 x g for 2 minutes. 18 S 12) Transfer the supernatant to the fresh tube containing 2 L of Solution IV. 13) Add 600 L of ice-cold 2-propanol to each tube, vortex briefly but thoroughly, then spin-down. Incubate the tube at -20oC for 20 minutes. Centrifuge the tube at 12,000 x g Analysis of isolated RNA with Bioanalyzer. for 10 minutes. Average of the RNA Quantity (n=2) 14) Wash the pellet 2 times with 0.5 mL of ice-cold 70% Antibody RNA (ng) Ethanol and dry up the pellet for 5-15 minutes. 15) Dissolve the pellets in nuclease-free water. Normal Rabbit IgG 76.5 16) RNA was quantified with NanoDrop (Thermo Fisher anti-SLBP 290.4 Scientific Inc.) and the RNA quality was analyzed with Total RNA 525585.0 Bioanalyzer (Agilent Technologies, Inc.). PROTOCOLS: (Positive control for RNP Immunoprecipitation; 293T) RNP Immunoprecipitation 123456789 Some buffers and reagents are included in the RIP-Assay Kit kDa (code. RN1001). Please also refer to the protocol packaged in the RIP-Assay Kit. 75 50 [Material Preparation] SLBP 1. Lysis Buffer (+) 37 Before using the Lysis Buffer, protease inhibitors, RNase inhibitors, and DTT are added to the Lysis Buffer at the appropriate concentration. 2. Wash Buffer (+) 25 Before using the Wash Buffer, DTT is added to the Wash Buffer at the appropriate concentration. Western blot analysis of SLBP expression in 293T (1), HeLa (2), K562 Protocol (3), Jurkat (4), MCF7 (5), NIH/3T3 (6), 1) Wash 2.1 x 107 cells 4 times with PBS and resuspend WR19L (7), Rat1 (8) and CHO (9) using them with 500 L of ice-cold Lysis Buffer (+) containing RN045P. appropriate protease inhibitors, RNase inhibitors, and DTT. Vortex thoroughly, then incubate it on ice for 10 SDS-PAGE & Western Blotting minutes. 1) Wash 1 x 107 cells 3 times with PBS and suspend them in 2) Centrifuge the tube at 12,000 x g for 5 minutes at 4oC and 1 mL of Laemmli’s sample buffer. transfer the supernatant to another tube. 2) Boil the samples for 2 minutes and centrifuge. Load 10 L 3) Add 25 L of 50% protein A agarose beads slurry of sample per lane on a 1-mm-thick SDS-polyacrylamide resuspended in Lysis Buffer (+) into the supernatant. gel and carry out electrophoresis. Incubate it at 4oC with rotating for 1 hour. 3) Blot the protein to a polyvinylidene difluoride (PVDF) 4) Centrifuge the tube at 2,000 x g for 1 minute at 4oC and membrane at 1 mA/cm2 for 1 hour in a semi-dry transfer transfer the supernatant to another fresh tube (precleared system (Transfer Buffer: 25 mM Tris, 190 mM glycine, sample). 20% MeOH). See the manufacturer's manual for precise 5) Mix 25 L of 50% protein A agarose beads slurry transfer procedure. RN045P Page 3 of 4 4) To reduce nonspecific binding, soak the membrane in 5% transfer the supernatant to another tube (precleared skimmed milk (in PBS, pH 7.2) for 1 hour at room sample). temperature, or overnight at 4oC. 5) Mix 25 L of 50% protein A agarose beads slurry 5) Incubate the membrane with primary antibody diluted resuspended in PBS with Normal Rabbit IgG (RIP-Assay with PBS, pH 7.2 containing 1% skimmed milk as Kit) or anti-SLBP antibody at the concentration suggested suggested in the APPLICATIONS for 1 hour at room in the APPLICATIONS, and then add 1 mL of Wash temperature. (The concentration of antibody will depend buffer into each tube. Incubate with gently agitation for 1 on the conditions.) hour at 4oC. 6) Wash the membrane with PBS-T [0.05% Tween-20 in 6) Wash the beads once with ice-cold Lysis Buffer PBS] (5 minutes x 3 times). (centrifuge the tube at 2,000 x g for 1 minute). Carefully 7) Incubate the membrane with the 1:5,000 HRP-conjugated discard the supernatant using a pipettor without disturbing anti-rabbit IgG (MBL; code no. 458) diluted with 1% the beads. skimmed milk (in PBS, pH 7.2) for 1 hour at room 7) Add 500 L of cell lysate (precleared sample of step 4), temperature. then incubate with gentle agitation for 3 hours at 4oC 8) Wash the membrane with PBS-T (5 minutes x 3 times). 8) Wash the beads 4 times with Wash Buffer (centrifuge the 9) Wipe excess buffer off the membrane, and incubate tube at 2,000 x g for 1 minute). membrane with an appropriate chemiluminescence 9) Resuspend the beads in 20 L of Laemmli’s sample buffer, reagent for 1 minute. boil for 3-5 minutes, and centrifuge for 5 minutes. Use 10 10) Remove extra reagent from the membrane by dabbing L/lane for the SDS-PAGE analysis. with a paper towel, and seal it in plastic wrap.
Load more

Recommended publications
  • Symplekin and Multiple Other Polyadenylation Factors Participate in 3 -End Maturation of Histone Mrnas
    Downloaded from genesdev.cshlp.org on September 27, 2021 - Published by Cold Spring Harbor Laboratory Press Symplekin and multiple other polyadenylation factors participate in 3؅-end maturation of histone mRNAs Nikolay G. Kolev and Joan A. Steitz1 Howard Hughes Medical Institute, Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06536, USA .Most metazoan messenger RNAs encoding histones are cleaved, but not polyadenylated at their 3؅ ends Processing in mammalian cell extracts requires the U7 small nuclear ribonucleoprotein (U7 snRNP) and an unidentified heat-labile factor (HLF). We describe the identification of a heat-sensitive protein complex whose integrity is required for histone pre-mRNA cleavage. It includes all five subunits of the cleavage and polyadenylation specificity factor (CPSF), two subunits of the cleavage stimulation factor (CstF), and symplekin. Reconstitution experiments reveal that symplekin, previously shown to be necessary for cytoplasmic poly(A) tail elongation and translational activation of mRNAs during Xenopus oocyte maturation, is the essential heat-labile component. Thus, a common molecular machinery contributes to the nuclear maturation of mRNAs both lacking and possessing poly(A), as well as to cytoplasmic poly(A) tail elongation. [Keywords: Symplekin; polyadenylation; 3Ј-end processing; U7 snRNP; histone mRNA; Cajal body] Received September 1, 2005; revised version accepted September 12, 2005. During the S phase of the cell cycle, histone mRNA lev- unique component of the U7-specific Sm core, in which els are up-regulated to meet the need for histones to the spliceosomal SmD1 and SmD2 proteins are replaced package newly synthesized DNA. Increased transcrip- by Lsm10 and Lsm11 (Pillai et al.
    [Show full text]
  • The Role of Nuclear Bodies in Gene Expression and Disease
    Biology 2013, 2, 976-1033; doi:10.3390/biology2030976 OPEN ACCESS biology ISSN 2079-7737 www.mdpi.com/journal/biology Review The Role of Nuclear Bodies in Gene Expression and Disease Marie Morimoto and Cornelius F. Boerkoel * Department of Medical Genetics, Child & Family Research Institute, University of British Columbia, Vancouver, BC V5Z 4H4, Canada; E-Mail: [email protected] * Author to whom correspondence should be addressed; E-Mail: [email protected]; Tel.: +1-604-875-2157; Fax: +1-604-875-2376. Received: 15 May 2013; in revised form: 13 June 2013 / Accepted: 20 June 2013 / Published: 9 July 2013 Abstract: This review summarizes the current understanding of the role of nuclear bodies in regulating gene expression. The compartmentalization of cellular processes, such as ribosome biogenesis, RNA processing, cellular response to stress, transcription, modification and assembly of spliceosomal snRNPs, histone gene synthesis and nuclear RNA retention, has significant implications for gene regulation. These functional nuclear domains include the nucleolus, nuclear speckle, nuclear stress body, transcription factory, Cajal body, Gemini of Cajal body, histone locus body and paraspeckle. We herein review the roles of nuclear bodies in regulating gene expression and their relation to human health and disease. Keywords: nuclear bodies; transcription; gene expression; genome organization 1. Introduction Gene expression is a multistep process that is vital for the development, adaptation and survival of all living organisms. Regulation of gene expression occurs at the level of transcription, RNA processing, RNA export, translation and protein degradation [1±3]. The nucleus has the ability to modulate gene expression at each of these levels. How the nucleus executes this regulation is gradually being dissected.
    [Show full text]
  • INT6 Interacts with MIF4GD/SLIP1 and Is Necessary for Efficient Histone Mrna Translation
    Downloaded from rnajournal.cshlp.org on September 26, 2021 - Published by Cold Spring Harbor Laboratory Press INT6 interacts with MIF4GD/SLIP1 and is necessary for efficient histone mRNA translation JULIA NEUSIEDLER,1,3,4 VINCENT MOCQUET,1,3 TARAN LIMOUSIN,2 THEOPHILE OHLMANN,2 CHRISTELLE MORRIS,1 and PIERRE JALINOT1,5 1Laboratoire de Biologie Mole´culaire de la Cellule, Unite´ Mixte de Recherche 5239, Centre National de la Recherche Scientifique, Ecole Normale Supe´rieure de Lyon, 69364 Lyon cedex 07, France 2Virologie Humaine, Unite´ 758, Institut National de la Sante´ et de la Recherche Me´dicale, Ecole Normale Supe´rieure de Lyon, 69364 Lyon cedex 07, France ABSTRACT The INT6/EIF3E protein has been implicated in mouse and human breast carcinogenesis. This subunit of the eIF3 translation initiation factor that includes a PCI domain exhibits specific features such as presence in the nucleus and ability to interact with other important cellular protein complexes like the 26S proteasome and the COP9 signalosome. It has been previously shown that INT6 was not essential for bulk translation, and this protein is considered to regulate expression of specific mRNAs. Based on the results of a two-hybrid screen performed with INT6 as bait, we characterize in this article the MIF4GD/SLIP1 protein as an interactor of this eIF3 subunit. MIF4GD was previously shown to associate with SLBP, which binds the stem–loop located at the 39 end of the histone mRNAs, and to be necessary for efficient translation of these cell cycle–regulated mRNAs that lack a poly(A) tail. In line with the interaction of both proteins, we show using the RNA interference approach that INT6 is also essential to S-phase histone mRNA translation.
    [Show full text]
  • NMR Structure and Dynamics of the RNA-Binding Site for the Histone Mrna Stem-Loop Binding Protein
    Downloaded from rnajournal.cshlp.org on September 30, 2021 - Published by Cold Spring Harbor Laboratory Press RNA (2002), 8:83–96+ Cambridge University Press+ Printed in the USA+ Copyright © 2002 RNA Society+ DOI: 10+1017+S1355838201013863 NMR structure and dynamics of the RNA-binding site for the histone mRNA stem-loop binding protein ERIC S. DEJONG,1 WILLIAM F. MARZLUFF,2 and EDWARD P. NIKONOWICZ1 1 Department of Biochemistry and Cell Biology, Rice University, Houston, Texas 77251-1892, USA 2 Program in Molecular Biology and Biotechnology, Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill, North Carolina 27599, USA ABSTRACT The 39 end of replication-dependent histone mRNAs terminate in a conserved sequence containing a stem-loop. This 26-nt sequence is the binding site for a protein, stem-loop binding protein (SLBP), that is involved in multiple aspects of histone mRNA metabolism and regulation. We have determined the structure of the 26-nt sequence by multidimen- sional NMR spectroscopy. There is a 16-nt stem-loop motif, with a conserved 6-bp stem and a 4-nt loop. The loop is closed by a conserved U•A base pair that terminates the canonical A-form stem. The pyrimidine-rich 4-nt loop, UUUC, is well organized with the three uridines stacking on the helix, and the fourth base extending across the major groove into the solvent. The flanking nucleotides at the base of the hairpin stem do not assume a unique conformation, despite the fact that the 59 flanking nucleotides are a critical component of the SLBP binding site.
    [Show full text]
  • A Multiprotein Occupancy Map of the Mrnp on the 3 End of Histone
    Downloaded from rnajournal.cshlp.org on October 6, 2021 - Published by Cold Spring Harbor Laboratory Press A multiprotein occupancy map of the mRNP on the 3′ end of histone mRNAs LIONEL BROOKS III,1 SHAWN M. LYONS,2 J. MATTHEW MAHONEY,1 JOSHUA D. WELCH,3 ZHONGLE LIU,1 WILLIAM F. MARZLUFF,2 and MICHAEL L. WHITFIELD1 1Department of Genetics, Dartmouth Geisel School of Medicine, Hanover, New Hampshire 03755, USA 2Integrative Program for Biological and Genome Sciences, University of North Carolina, Chapel Hill, North Carolina 27599, USA 3Department of Computer Science, University of North Carolina, Chapel Hill, North Carolina 27599, USA ABSTRACT The animal replication-dependent (RD) histone mRNAs are coordinately regulated with chromosome replication. The RD-histone mRNAs are the only known cellular mRNAs that are not polyadenylated. Instead, the mature transcripts end in a conserved stem– loop (SL) structure. This SL structure interacts with the stem–loop binding protein (SLBP), which is involved in all aspects of RD- histone mRNA metabolism. We used several genomic methods, including high-throughput sequencing of cross-linked immunoprecipitate (HITS-CLIP) to analyze the RNA-binding landscape of SLBP. SLBP was not bound to any RNAs other than histone mRNAs. We performed bioinformatic analyses of the HITS-CLIP data that included (i) clustering genes by sequencing read coverage using CVCA, (ii) mapping the bound RNA fragment termini, and (iii) mapping cross-linking induced mutation sites (CIMS) using CLIP-PyL software. These analyses allowed us to identify specific sites of molecular contact between SLBP and its RD-histone mRNA ligands. We performed in vitro crosslinking assays to refine the CIMS mapping and found that uracils one and three in the loop of the histone mRNA SL preferentially crosslink to SLBP, whereas uracil two in the loop preferentially crosslinks to a separate component, likely the 3′hExo.
    [Show full text]
  • C. Elegans Cdl-1 Encodes a SLBP
    Development 129, 187-196 (2002) 187 Printed in Great Britain © The Company of Biologists Limited 2002 DEV5960 The stem-loop binding protein CDL-1 is required for chromosome condensation, progression of cell death and morphogenesis in Caenorhabditis elegans Yuki Kodama1, Joel H. Rothman2, Asako Sugimoto1,3,4,* and Masayuki Yamamoto1 1Department of Biophysics and Biochemistry, Graduate School of Science, University of Tokyo, Bunkyo, 113-0032, Japan 2Neuroscience Research Institute, University of California-Santa Barbara, Santa Barbara, CA 93106, USA 3PRESTO, Japan Science and Technology Corporation, Kawaguchi, 332-0012, Japan 4Laboratory for Developmental Genomics, RIKEN Center for Developmental Biology, Kobe, 650-0047 Japan *Author for correspondence [e-mail: [email protected], [email protected] (from April 2002)] Accepted 5 October 2001 SUMMARY Histones play important roles not only in the structural loop structures in the 3′-UTR of C. elegans core histone changes of chromatin but also in regulating gene mRNAs, and the mutant forms of this protein show reduced expression. Expression of histones is partly regulated binding activities. A decrease in the amount of core histone post-transcriptionally by the stem-loop binding protein proteins phenocopied the cdl-1 mutant embryos, suggesting (SLBP)/hairpin binding protein (HBP). We report the that CDL-1 contributes to the proper expression of core developmental function of CDL-1, the C. elegans histone proteins. We propose that loss-of-function of cdl-1 homologue of SLBP/HBP. In the C. elegans cdl-1 mutants, causes aberrant chromatin structure, which affects the cell cell corpses resulting from programmed cell death appear cycle and cell death, as well as transcription of genes later and persist much longer than those in the wild type.
    [Show full text]
  • Structural and Biochemical Studies of SLIP1–SLBP Identify DBP5 And
    7960–7971 Nucleic Acids Research, 2013, Vol. 41, No. 16 Published online 26 June 2013 doi:10.1093/nar/gkt558 Structural and biochemical studies of SLIP1–SLBP identify DBP5 and eIF3g as SLIP1-binding proteins Holger von Moeller1, Rachel Lerner2, Adele Ricciardi2, Claire Basquin1, William F. Marzluff2 and Elena Conti1,* 1Structural Cell Biology Department, Max Planck Institute of Biochemistry, Munich, D-82152 Germany and 2Program in Molecular Biology and Biotechnology, University of North Carolina, Chapel Hill, NC 27599, USA Received March 5, 2013; Revised May 27, 2013; Accepted May 31, 2013 ABSTRACT integral part of the mature messenger ribonucleoprotein particle (mRNP) that is exported to the cytoplasm. In the Downloaded from In metazoans, replication-dependent histone cytoplasm, PABP interacts with the eukaryotic initiation mRNAs end in a stem-loop structure instead of the factor 4G (eIF4G), which in turn is recruited to the 50 end poly(A) tail characteristic of all other mature mRNAs. of the transcript (2,3). The network of interactions con- This specialized 30 end is bound by stem-loop necting the 30 and 50 ends of an mRNA promotes transla- binding protein (SLBP), a protein that participates tion initiation and hampers degradation [reviewed in (4)]. http://nar.oxfordjournals.org/ in the nuclear export and translation of histone Mammalian mRNAs transcribed from replication-de- mRNAs. The translational activity of SLBP is pendent histone genes are the exception to the norm. mediated by interaction with SLIP1, a middle Histones are massively produced during S-phase to domain of initiation factor 4G (MIF4G)-like protein assemble the newly synthesized genomic DNA into chro- that connects to translation initiation.
    [Show full text]
  • Thyroid Hormone Increases Bulk Histones Expression by Enhancing Translational Efficiency
    ORIGINAL RESEARCH Thyroid Hormone Increases Bulk Histones Expression by Enhancing Translational Efficiency Alberto Zambrano,* Verónica García-Carpizo,* Raquel Villamuera, and Ana Aranda Instituto de Investigaciones Biomédicas “Alberto Sols,” Consejo Superior de Investigaciones Científicas and Universidad Autónoma de Madrid, 28029 Madrid, Spain Downloaded from https://academic.oup.com/mend/article/29/1/68/2556825 by guest on 02 October 2021 The expression of canonical histones is normally coupled to DNA synthesis during the S phase of the cell cycle. Replication-dependent histone mRNAs do not contain a poly(A) tail at their 3Ј terminus, but instead possess a stem-loop motif, the binding site for the stem-loop binding protein (SLBP), which regulates mRNA processing, stability, and relocation to polysomes. Here we show that the thyroid hormone can increase the levels of canonical histones independent of DNA replication. Incubation of mouse embryonic fibroblasts with T3 increases the total levels of histones, and expression of the thyroid hormone receptor ␤ induces a further increase. This is not restricted to mouse embryonic fibroblasts, because T3 also raises histone expression in other cell lines. T3 does not increase histone mRNA or SLBP levels, suggesting that T3 regulates histone expression by a posttranscriptional mech- anism. Indeed, T3 enhanced translational efficiency, inducing relocation of histone mRNA to heavy polysomes. Increased translation was associated with augmented transcription of the eukaryotic ␥ translation initiation factor 4 2 (EIF4G2). T3 induced EIF4G2 protein and mRNA levels and the thyroid hormone receptor bound to the promoter region of the Eif4g2 gene. Induction of EIF4G2 was essential for T3-dependent histone induction, because depletion of this factor abolished histone increase.
    [Show full text]
  • Poster After
    The Interaction of UPF1 with the Stem-loop Binding Protein is Critical for Initiation of Histone mRNA Degradation at the end of S-phase Stacie Meaux1, Akio Yamashita2 and William Marzluff1 1Program in Molecular Biology and Biotechnology, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599 2Department of Molecular Biology, Yokohama City University School of Medicine, Yokohama, Japan 232-0024 Abstract UPF1 Helicase Mutants Bind to SLBP UPF1 truncations are Dominant Negative for Histone mRNA Decay. Histone expression is a highly regulated process that is coordinated with DNA synthesis during the cell cycle. Histone mRNA levels remain at No a low basal level throughout most of the life of a cell but increase about 30-fold at the beginning of S phase. In vertebrates, this increase is a A UPF1 (WT) (K498A) (R843C) B plasmid K498A R843C Left: HeLa cells expressing the in- result of increased transcription and more efficient 3’ end processing. At the end of S-phase, histone mRNA levels decrease back to their basal dicated proteins were treated with level due to rapid degradation of the mRNAs. Degradation of histone mRNAs at the end of S phase (or when DNA synthesis is inhibited in None HA-UPF11-984 215-1129 HU, and RNA was isolated at the 0 20 40 60 80 min. S-phase cells by treatment with hydroxyurea (HU)) requires the normal mRNA degradation machinery. Degradation of histone mRNAs is initi- 5% IN PD 5% IN PD 5% IN PD UPF1 indicated times. Northern blots HA-UPF1 HeLa H2a ated by oligouridylation of their 3’ ends, binding of Lsm1-7 to the oligo(U) tail, followed by degradation from their 5’ ends by decapping and the HeLa were probed for the H2aa3 (H2a) 7SK 1000 exoribonuclease XRN1 and from their 3’ ends by the exosome complex (see Fig.1 and [1]).
    [Show full text]
  • A Region of Slbp Distinct from the Histone Mrna Binding and Processing Domains Is Essential for Deposition of Histone Mrna in the Oocyte
    A REGION OF SLBP DISTINCT FROM THE HISTONE MRNA BINDING AND PROCESSING DOMAINS IS ESSENTIAL FOR DEPOSITION OF HISTONE MRNA IN THE OOCYTE Jennifer M. Potter-Birriel A dissertation submitted to the faculty at the University of North Carolina at Chapel Hill in particial fulfillment of the requirements for the degree of Doctor of Philosophy in the Curriculum of Genetics and Molecular Biology. Chapel Hill 2020 Approved by: William F. Marzluff Robert J. Duronio Jill Dowen Zbig Dominski Cyrus Vaziri © 2020 Jennifer M. Potter-Birriel ALL RIGHTS RESERVED ii ABSTRACT Jennifer M. Potter-Birriel: A region of Drosophila SLBP distinct from the histone pre-mRNA binding and processing domains is essential for deposition of histone mRNA in the oocyte (Under the direction of Bill Marzluff) Metazoan histone mRNAs are the only eukaryotic mRNAs that are not polyadenylated. Instead they end in a 3’end Stemloop (SL). Processing of the histones pre-mRNAs is accomplished by an endonucleolytic cleavage after the SL. The stem loop binding protein (SLBP) binds to the SL, and SLBP is a key factor in all steps of the life cycle of histone mRNAs. We are studying the role of SLBP in Drosophila melanogaster in vivo. In Drosophila each histone gene contains a cryptic polyA site after the histone processing site, and when histone pre- mRNA processing is defective histone mRNAs are polyadenylated. Using FLY-CRISPRCas9 we obtained a 11 deletion (SLBP∆11) null mutant and a 30 nucleotide deletion (SLBP∆30) in the N-terminal domain (NTD) of SLBP. The 30nt deletion removed 10aa from the N-terminal domain of SLBP in a region of unknown function distinct from the processing domain.
    [Show full text]
  • Molecular Mechanisms for the Regulation of Histone Mrna Stem-Loop–Binding Protein by Phosphorylation
    Molecular mechanisms for the regulation of histone PNAS PLUS mRNA stem-loop–binding protein by phosphorylation Jun Zhanga, Dazhi Tanb, Eugene F. DeRosea, Lalith Pereraa, Zbigniew Dominskic,d, William F. Marzluffc,d,1, Liang Tongb,1, and Traci M. Tanaka Halla,1 aLaboratory of Structural Biology, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC 27709; bDepartment of Biological Sciences, Columbia University, New York, NY 10027; cDepartment of Biochemistry and Biophysics and dIntegrative Program in Biological and Genome Sciences, University of North Carolina, Chapel Hill, NC 27599 Edited by Jennifer A. Doudna, University of California, Berkeley, CA, and approved June 19, 2014 (received for review April 8, 2014) Replication-dependent histone mRNAs end with a conserved stem the stem-loop RNA (18). On the other hand, how SLBP alone loop that is recognized by stem-loop–binding protein (SLBP). The interacts with the RNA or how this interaction might be affected minimal RNA-processing domain of SLBP is phosphorylated at an by phosphorylation of the TPNK motif is not known. internal threonine, and Drosophila SLBP (dSLBP) also is phosphor- The C-terminal region of dSLBP contains a motif, SNSDSDSD, ylated at four serines in its 18-aa C-terminal tail. We show that whose hyperphosphorylation is required for efficient processing phosphorylation of dSLBP increases RNA-binding affinity dramat- of histone pre-mRNA (15). Despite the similarity of hSLBP ically, and we use structural and biophysical analyses of dSLBP and and dSLBP RBDs (55% identical residues) and their ability to a crystal structure of human SLBP phosphorylated on the internal bind identical stem-loop RNA sequences, neither SLBP can threonine to understand the striking improvement in RNA binding.
    [Show full text]
  • Molecular Mechanisms for the Regulation of Histone Mrna Stem
    Molecular mechanisms for the regulation of histone PNAS PLUS mRNA stem-loop–binding protein by phosphorylation Jun Zhanga, Dazhi Tanb, Eugene F. DeRosea, Lalith Pereraa, Zbigniew Dominskic,d, William F. Marzluffc,d,1, Liang Tongb,1, and Traci M. Tanaka Halla,1 aLaboratory of Structural Biology, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC 27709; bDepartment of Biological Sciences, Columbia University, New York, NY 10027; cDepartment of Biochemistry and Biophysics and dIntegrative Program in Biological and Genome Sciences, University of North Carolina, Chapel Hill, NC 27599 Edited by Jennifer A. Doudna, University of California, Berkeley, CA, and approved June 19, 2014 (received for review April 8, 2014) Replication-dependent histone mRNAs end with a conserved stem the stem-loop RNA (18). On the other hand, how SLBP alone loop that is recognized by stem-loop–binding protein (SLBP). The interacts with the RNA or how this interaction might be affected minimal RNA-processing domain of SLBP is phosphorylated at an by phosphorylation of the TPNK motif is not known. internal threonine, and Drosophila SLBP (dSLBP) also is phosphor- The C-terminal region of dSLBP contains a motif, SNSDSDSD, ylated at four serines in its 18-aa C-terminal tail. We show that whose hyperphosphorylation is required for efficient processing phosphorylation of dSLBP increases RNA-binding affinity dramat- of histone pre-mRNA (15). Despite the similarity of hSLBP ically, and we use structural and biophysical analyses of dSLBP and and dSLBP RBDs (55% identical residues) and their ability to a crystal structure of human SLBP phosphorylated on the internal bind identical stem-loop RNA sequences, neither SLBP can threonine to understand the striking improvement in RNA binding.
    [Show full text]