Project description:Using high-throughput sequencing of histone mRNAs and degradation intermediates, we find that knockdown of TUT7 reduces both the uridylation at the 3’ end as well as uridylation pattern at the 3’ end, and only had a small effect on the uridylation in the stemloop uridylation of the major during histone mRNA degradation. Knockdown of 3’ hEXO also altered the uridylation of histone mRNAs, revealing a dynamic equilibrium between 3’ hEXO digestion and TUT7 uridylation, suggesting that TUT7 and 3’ hExo function together in trimming and uridylating histone mRNAs.
Project description:Metazoan histone mRNAs are the only cellular eucaryotic mRNAs that are not polyadenylated, ending instead in a conserved stemloop. SLBP is bound to the 3’ end of histone mRNA and is required for translation of histone mRNA. Their expression is tightly cell-cycle regulated. A major regulatory step is rapid degradation of histone mRNA at the end of S-phase or when DNA synthesis is inhibited in S-phase. 3’hExo, a 3’ to 5’ exonuclease, binds to the SLBP/SL complex, and trims histone mRNA to 3 nts after the stemloop. Together with a terminal uridyl transferase, 3’hExo maintains the length of the histone mRNA during S-phase. 3’hExo is essential for initiating histone mRNA degradation on polyribosomes, initiating degradation into the 3’ side of the stemloop. There is extensive uridylation of degradation intermediates in the 3’ side of the stem when histone mRNA is degraded. Here we knocked out TUT7 and 3’hExo and we show that both modification of histone mRNA during S-phase and degradation of histone mRNA involve the interaction of 3’hExo, and a specific TUTase, TENT3B (TUT7, CHHC6). Knockout of 3’hExo prevents initiation of 3’ to 5’ degradation, stabilizing histone mRNA, while knockout of TUT7 prevents uridylation of the mRNA degradation intermediates slowing the rate of degradation. In synchronized 3’hExo KO cells, histone mRNA degradation is delayed but the histone mRNA is degraded prior to mitosis by a different pathway.
Project description:Terminal uridylyl transferases (TUTs) function as integral regulators of microRNA (miRNA) biogenesis by modifying the end structure of precursor miRNA (pre-miRNA). Using biochemistry and deep sequencing techniques, we here investigate the mechanism how human TUT7 recognizes and uridylates pre-miRNAs. We show that the overhang of a pre-miRNA is the key structural element that TUT7 and its paralogues, TUT4 and TUT2, recognize. For group II pre-miRNAs which have a 1 nt 3’ overhang, TUT7 restores the canonical end structure (2 nt 3’ overhang) by mono-uridylation, and thereby promotes miRNA biogenesis. Interestingly, once the 3’ end is receded into the stem (3’ trimmed pre-miRNAs such as Ago-cleaved-pre-miRNA), TUT7 effectively generates an oligo-U tail that consequently leads to degradation. Our single-molecule study further suggests that a distributive mode is employed for both pathways, but the overhang length determines the frequency of TUT7-RNA interaction. Our results explain how TUT7 and TUT4 differentiate pre-miRNA species and reveal a role for TUT7 and TUT4 in the oligo-uridylation and removal of defective pre-miRNAs. HeLa cells were knocked down of control or TUT2/4/7, then total RNAs were prepared for RNA-seq
Project description:Terminal uridylyl transferases (TUTs) function as integral regulators of microRNA (miRNA) biogenesis by modifying the end structure of precursor miRNA (pre-miRNA). Using biochemistry and deep sequencing techniques, we here investigate the mechanism how human TUT7 recognizes and uridylates pre-miRNAs. We show that the overhang of a pre-miRNA is the key structural element that TUT7 and its paralogues, TUT4 and TUT2, recognize. For group II pre-miRNAs which have a 1 nt 3’ overhang, TUT7 restores the canonical end structure (2 nt 3’ overhang) by mono-uridylation, and thereby promotes miRNA biogenesis. Interestingly, once the 3’ end is receded into the stem (3’ trimmed pre-miRNAs such as Ago-cleaved-pre-miRNA), TUT7 effectively generates an oligo-U tail that consequently leads to degradation. Our single-molecule study further suggests that a distributive mode is employed for both pathways, but the overhang length determines the frequency of TUT7-RNA interaction. Our results explain how TUT7 and TUT4 differentiate pre-miRNA species and reveal a role for TUT7 and TUT4 in the oligo-uridylation and removal of defective pre-miRNAs.
Project description:Uridylation occurs pervasively on mRNAs in mammals, yet its mechanism and significance remain unknown. Here we identify TUT4 and TUT7 (also known as ZCCHC11 and ZCCHC6, respectively) as the enzymes that uridylate mRNAs. Uridylation readily occurs on deadenylated mRNAs that are not associated with poly(A) binding protein (PABPC1) in cells. Consistently, purified TUT4 and TUT7 (TUT4/7) selectively uridylate RNAs with short A tails (< ~25 nt) while PABPC1 antagonizes uridylation of polyadenylated mRNAs in vitro. In cells depleted of TUT4/7, the vast majority of mRNAs lose the U tails, and their half-lives are extended. Suppression of mRNA decay factors leads to the accumulation of uridylated mRNAs. In line with this, microRNA induces uridylation of its targets, and TUT4/7 is required for enhanced decay of microRNA targets. Our study explains the mechanism underlying selective uridylation of deadenylated mRNAs, and demonstrates a fundamental role of the U tail as a molecular mark for global mRNA decay. Thirteen separate sets of TAIL-seq experiments were performed. Each set includes a negative control for transfection, immunoprecipitation, or knockout cell generation. Experimental samples were treated with various conditions including siRNA transfection, transdominant negative protein expression, TALEN-based gene knockout, or immunoprecipitation. The 'README-TAIL-seq.txt' include detailed information about structure of seq entries in FASTQ files and of processed data for 3' end modifications.
Project description:Uridylation occurs pervasively on mRNAs in mammals, yet its mechanism and significance remain unknown. Here we identify TUT4 and TUT7 (also known as ZCCHC11 and ZCCHC6, respectively) as the enzymes that uridylate mRNAs. Uridylation readily occurs on deadenylated mRNAs that are not associated with poly(A) binding protein (PABPC1) in cells. Consistently, purified TUT4 and TUT7 (TUT4/7) selectively uridylate RNAs with short A tails (< ~25 nt) while PABPC1 antagonizes uridylation of polyadenylated mRNAs in vitro. In cells depleted of TUT4/7, the vast majority of mRNAs lose the U tails, and their half-lives are extended. Suppression of mRNA decay factors leads to the accumulation of uridylated mRNAs. In line with this, microRNA induces uridylation of its targets, and TUT4/7 is required for enhanced decay of microRNA targets. Our study explains the mechanism underlying selective uridylation of deadenylated mRNAs, and demonstrates a fundamental role of the U tail as a molecular mark for global mRNA decay. HeLa cells were knocked down of control or TUT4/7, then total RNAs were prepared for RNA-seq on 0, 1, 2, 4h after actinomycin D treatment. The whole processes of experiments were repeated two times.