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]). Previous work from our lab has shown that UPF1 5% input GST-SLBP 5% input GST-SLBP 5% input GST-SLBP End. SLBP or 7SK RNAs to control for loading. interacts with SLBP after HU treatment and that the helicase activity of UPF1 is required for histone mRNA decay [2]. Hela Top Right: Western blot showing HA-UPF1 H2a HA-UPF1 HA-UPF1 the expression levels of the indicat- I have shown that UPF1 and SLBP interact directly and have identified the binding regions of both of these proteins. UPF1 binds to SLBP near HA-UPF1 7SK its RNA binding domain, while SLBP binds UPF1 at its internal, helicase domain. Interestingly, removal of the N-terminal Zn-finger domain and 215-1129 ed proteins. Bottom Right: Decay C-terminal [ST]-Q motifs of UPF1 are dominant negative for histone mRNA decay. Furthermore, the C-terminal SQ motifs of UPF1 are re- 5% IN PD H2a 100 1-984 plots of the Northern blots repre- Avg % Input 1.2 1.3 1.1 HA-UPF1 1-984 quired for binding to SLBP. I have also discovered that SMG1, SMG5 and SMG7 but not SMG6 are involved in histone mRNA degradation. 7SK senting averages of at least 3 ex- SLBP- periments. SBP SLBP-SBP 1. Mullen,T.E. and Marzluff,W.F., Degradation of histone mRNA requires oligouridylation followed by decapping and simultaneous degradation H2a End. SLBP 215-1129 of the mRNA both 5' to 3' and 3' to 5', Genes Dev., 22 (2008) 50-65. 7SK 10 2. Kaygun, H. and Marzluff, W.F., Regulated degradation of replication-dependent histone mRNAs requires both ATR and Upf1, Nat. Struc. 0 10 20 30 40 50 60 70 80 90 A. In vitro transcribed UPF1 (and helicase mutants) were pulled down with GST-SLBP. All mutants bind as well as the WT protein. B. Stable and Mol. Biol., 12(9) (2005) 794-800. cell lines expressing the SLBP-SBP protein as well as HA-UPF1 or either of the helicase mutants were tested for in vivo binding. The UPF1 helicase mutants bind SLBP-SBP as well as WT. SMG1 is Involved in Histone mRNA Decay but UPF1 Binds at residues 92-127 of SLBP Does not Affect Binding of UPF1 to SLBP. Histone mRNAs are Regulated with the Cell Cycle LSM4 Binding A. HeLa cells were treated with the Protein UPF1/RF3 TUT3 Binding 120 siRNA: C2 SMG1 A A C2 B indicated siRNAs and then with Degradation Binding 100 A B Cyclin E RNA Processing SMG1 HU. RNA was isolated at the indi- 80 Upf1 cated time, and Northern blots SLBP 0 3 6 9 Hours Translation Binding RBD P 5% IN PD 5% IN PD were probed for histone mRNAs. HeLa SLBP-SBP P P 60 Histone Coding Region P Performed by T. Mullen and K. 40 UPF1 Lakerman. B. SLBP-SBP cells SLBP-SBP End. SLBP 20 SLBP-SBP were exposed to siRNA against 35 mRNA Remaining End. SLBP 1 60 64 78 82 97 100 125 171 174 197 217 270 0 SMG1 and then incubated with Completion of 0 20 45 End. SLBP DNA Synthesis + Stem-loop streptavadin beads. Western blots 30 Time (minutes) GST-SLBP Proteins 100 ng 200 ng 500 ng were probed for UPF1 and SLBP Inhibition of DNA B siRNA: C2 hSMG-1 - HU + HU (top) or for SMG1 (bottom). Synthesis 20 C2 ATR hSMG1 C2 UPF1 5% IN 5% IN 5% IN 5% IN GST- GST- SLBP GST- SLBP SLBP anti-hSMG1 GST- SLBP FL 1-68 92-270 5% IN 69-270 GST 1-127 Histone mRNA 1% IN AP 1% IN AP 128-270 anti-SMG1 anti-UPF1 SLBP Protein Relative Levels End. UPF1 HA-UPF1 10 HA-UPF1 SLBP-SBP %PD 4.3 2.4 1.7 SMG7/5 but not SMG6 Associates with SLBP GST-SLBP HeL SLBP-SB 1 End. SLBP A B M G S G M 100 GST-SLBP IN -tRNA +tRNA 0 20 40 60 80 min AP Hela 10% 10% AP HA-UPF1 HeLa H2a SMG6 shRNA A. Top: The stem-loop binding protein (SLBP) binds to a conserved stem-loop sequence at the 3’ end of histone mRNAs, which lack a poly(A) anti-UPF1 7SK tail. SLBP is required for all steps of histone mRNA metabolism. Bottom: At the beginning of S-phase, histone mRNA levels (red line) increase GST %PD 4.2 4.5 about thirty-fold due to increased transcription and more efficient 3’ end processing. At the end of S-phase, histone transcripts are rapidly de- SMG6 H2a GST-SLBP anti-SMG6 graded. At the beginning of S-phase, the stem-loop binding protein (SLBP) protein (blue line) increases about 20-fold and is degraded at the 7SK 10 end of S phase. B. SLBP-SBP functions like endogenous SLBP. Top Left: SLBP-SBP is expressed in HeLa cells but at levels that are lower anti-UPF1 than endogenous. Top Right: Double thymidine block followed by release into S-phase of SLBP-SBP cells shows that SLBP-SBP is regulated A. Graphical representation of SLBP, its known functional domains, and the binding domains of its known binding partners. RBD = RNA SMG6 shRNA like endogenous SLBP. Bottom: SLBP-SBP recruits UPF1 after treatment with HU. anti-SMG7 Binding Domain (B) Left: GST pull-downs using purified GST, GST-SLBP and truncated versions of GST-SLBP and radiolabeled HA-UPF1. + - Top Right: GST pull-downs using full-length GST-SLBP and radiolabeled HA-UPF1 with the indicated amounts of biotinylated histone anti-SMG5 The Current Model for Histone mRNA Decay 1 anti-SLBP 1. Efficient translation of a histone 0 20 40 60 80 100 Histone mRNA SLBP Binds within the Helicase Domain of UPF1, but EIF4G Degradation is Initiated mRNA occurs due to circularization EIF4G EIF4E of the transcript by interactions be- SLIP1 3’hEXO Removal of the Zn-finger Domain Increases Binding A. Lysates from HeLa or SLBP-SBP cells were incubated with streptavadin beads, and western blots were performed using the indicated an- AUG EIF4E SLIP1 SLBP 3’hEXO tween SLBP, SLIP1, EIF4G and ERF3a AUG tibodies. (B) HeLa cells were infected with SMG6 shRNA Lentivirus and then treated with HU. RNA was isolated at the indicated time points, SLBP* EIF4E. SLBP could also make and Removal of the SQ motifs Decreases Binding. ERF1 UPF1* ERF3a and Northern blots were probed for the indicated RNAs. Bottom: Western blot for SMG6 of the indicated cells. 1. UAA ERF1 translation more efficient by bind- eRF3 binding (A) Graphical representation of UPF1 UAA ing translation release factors. 2. UPF2/STAU1 binding and its interaction domains for known 2. A SLBP binding When DNA synthesis is inhibited, SMG6 SMG5/7 binding partners. (B) GST pull-downs The Role of UPF1 in Histone mRNA Decay. CBP80 binding LSM4 binding signals for the initiation of histone binding binding using GST or GST-SLBP and radiola- T28 K498 R843 S1096 TUT mRNA degradation are generated C beled HA-UPF1 (full-length or truncat- Histone LSM1- in the nucleus. Translation termi- ed). (C) Streptavadin beads were incu- mRNA 7 nation of the histone transcript is TUT SLBP TuTase bated with lysates from HeLa cells ex- LSM1- compromised, possibly by modifi- U 7 U 3’hEXO [ST]-Q pressing the indicated plasmids. West- U U Zn-finger ATP-binding U UU UU U SLBP U SLBP cation of SLBP, and UPF1 is re- SLBP U SLBP U EIF4G U 1 100 200 300 400 500 600 700 800 900 1000 1129 ern blots were probed with anti-HA and U SLBP U U U U UU U U UU U XRN1 AUG cruited to the mRNP. 3.