Use of CRISPR technology to investigate the mechanism of mRNA degradation Contact: Wenxia He1*, Christopher Erik Holmquist1, William F. Marzluff1,2 [email protected] * Presenting Author; 1Department of Biology; 2Department of Biochemistry, Integrated Program in Biological and Genome Sciences, University of North Carolina

Histone mRNA degradation KO of 3’hExo&TUT7 3’hExo&TUT7 KO Phenotype 3’hExo&TUT7 KO Rescue

Rescue of 3’hExo KO KO of 3’hExo A lentivirus (made an active(wt) and catalytically dead(cd) virus - express 3’hexo from doxycycline inducible 3’hExo promoter – puromycin resistant bactin

wt 3h13 3h13wt 3h13CD 0 20 40 0 20 40 0 20 40 0 20 40 HU (min) 11.4 histone mrna and slbp are cell-cycle regulated: histone mrna but not slbp are degraded when is inhibited TUT7 reduces 3’hExo KO abolish the degradation. TUT7 may reduces the degradation rate. KO of TUT7 TRANSLATING PABP CYTOPLASMIC AAAAAAAAAAAPABP AAA WT T73 T73R WT T73 T73R eIF3 Rescue of TUT7 TUT7 TUT4 40S A lentivirus plasmid- express 4E a TUT7 had been infected to the CAP TUT KO cells. TUT7 KO express 20-40 nts higher TUT4, but TUT7R(rescue) express less TUT4. eIF3 SLIP1 bactin bactin uridylation was reduced about 95% at the 3’ end of histone mRNAs in the TUT7 KO Sphase cells 40S 4E 23-50 m7GpppN SLBP nts 11.4 Deletion of SLBP exon2 Phenotype of SLBP mutation Abstract&Conclusion Histone mRNAs differ from all other cellular mRNAs in that they do not POLY A mRNA Northern blot end in a polyA tail, but end instead in a stemloop. Histone mRNAs are tightly cell-cycle regulated and most of the regulation in mammalian WT S10 S19 cells is postranscriptional. An important regulatory step is rapid

RNA Binding Domain 0 20 40 0 20 40 0 20 40 HU(min) changes in the rate of histone mRNA degradation to balance histone (Wang 1996) mRNA levels with the rate of DNA replication. The stem-loop at the 3’ 55-59 71-80 85-89 127 199 219 H2a end of histone mRNA binds SLBP and is the critical cis-element that The stemloop at the 3’ end is cis-element for regulated degradation of controls histone mRNA half-life. The SLBP-stem-loop RNA complex is histone mRNA. is required for histone mrna degradation involved in all steps of histone mRNA metabolism. SLBP-mediated gRNA1/2 replication-dependent histone mRNAs decay depends on translation Uridylation and EXON2 117NT and UPF1 recruitment to the 3’ UTR of histone mRNA when DNA Degradation of replication is inhibited. UPF1 recruitment results in activation of 3’hExo Histone mRNAAfter gRNA3/4 and TUTase 7 to initiate histone mRNA degradation. Using processing the crispr/cas9, we deleted exon2 (aa 14-50) of SLBP, on the N-terminal histone mRNA is 7SK and found the rate of degradation of histone mRNA became slower. trimmed by 3′hExo. If 11.4 11.4 trimming proceeds to Dr.Sutapa Chakrabarti’s lab (Free University of Berlin), have found that within 1–2 nt of the exon2 this region of SLBP directly binds Upf1, providing a mechanism to stem the mRNA is S10 S19 recruit Upf1 to histone mRNA. We postulate that Upf1 binds to the repaired by TUT7 to terminating ribosome and also to the N-terminal region of SLBP. This the proper length. When DNA replication results in activation of 3’hExo and TUT7 to initiate histone mRNA is inhibited, Upf1 is SLBP RT-PCR SLBP WB degradation. We have knocked out both TUT7 and 3’hExo and will recruited to the also determine the regions in these required for histone WT S10 S19 WT S10 S19 histone mRNA, and F R mRNA degradation. 3′hExo can degrade 1 2 3 4 5 6 7 8 into the stem. When Numbers are exon boundaries the nuclease stalls 1 17 55 88 108 154 205 227 end the mRNA is then uridylated and Acknowledgements degradation can resume. Marzluff Lab Sutapa Lab Band 2 likely alternative splicing of a small amount of cells with mutant slbp degrade histone mRNA more slowly 11.4 Band 1, slbp/SLBP(mutation) exon 1-3 in wild-type Band 2