Minju Ha

TUT4 in Concert with Lin28 Suppresses MicroRNA Biogenesis through Pre-MicroRNA Uridylation

As key regulators in cellular functions, microRNAs (miRNAs) themselves need to be tightly controlled. Lin28, a pluripotency factor, was reported to downregulate let-7 miRNA by inducing uridylation of let-7 precursor (pre-let-7). But the enzyme responsible for the uridylation remained unknown. Here we identify a noncanonical poly (A) polymerase, TUTase4 (TUT4), as the uridylyl transferase for pre-let-7. Lin28 recruits TUT4 to pre-let-7 by recognizing a tetra-nucleotide sequence motif (GGAG) in the terminal loop. TUT4 in turn adds an oligouridine tail to the pre-let-7, which blocks Dicer processing. Other miRNAs with the same sequence motif (miR-107, -143, and -200c) are regulated through the same mechanism. Knockdown of TUT4 and Lin28 reduces the level of stem cell markers, suggesting that they are required for stem cell maintenance. This study uncovers the role of TUT4 and Lin28 as specific suppressors of miRNA biogenesis, which has implications for stem cell research and cancer biology.

Short Structured RNAs with Low GC Content Are Selectively Lost during Extraction from a Small Number of Cells

Document S1. Supplemental Experimental Procedures and Figures S1 and S2xDownload (.31 MB ) Document S1. Supplemental Experimental Procedures and Figures S1 and S2

Uridylation by TUT4 and TUT7 Marks mRNA for Degradation

Uridylation occurs pervasively on mRNAs, yet its mechanism and significance remain unknown. By applying TAIL-seq, we identify TUT4 and TUT7 (TUT4/7), also known as ZCCHC11 and ZCCHC6, respectively, as mRNA uridylation enzymes. Uridylation readily occurs on deadenylated mRNAs in cells. Consistently, purified TUT4/7 selectively recognize and uridylate RNAs with short A-tails (less than ∼ 25 nt) in vitro. PABPC1 antagonizes uridylation of polyadenylated mRNAs, contributing to the specificity for short A-tails. In cells depleted of TUT4/7, the vast majority of mRNAs lose the oligo-U-tails, and their half-lives are extended. Suppression of mRNA decay factors leads to the accumulation of oligo-uridylated mRNAs. In line with this, microRNA induces uridylation of its targets, and TUT4/7 are required for enhanced decay of microRNA targets. Our study explains the mechanism underlying selective uridylation of deadenylated mRNAs and demonstrates a fundamental role of oligo-U-tail as a molecular mark for global mRNA decay.

TUT7 controls the fate of precursor microRNAs by using three different uridylation mechanisms

Terminal uridylyl transferases (TUTs) function as integral regulators of microRNA (miRNA) biogenesis. Using biochemistry, single‐molecule, and deep sequencing techniques, we here investigate the mechanism by which human TUT7 (also known as ZCCHC6) recognizes and uridylates precursor miRNAs (pre‐miRNAs) in the absence of Lin28. We find that the overhang of a pre‐miRNA is the key structural element that is recognized by TUT7 and its paralogues, TUT4 (ZCCHC11) and TUT2 (GLD2/PAPD4). For group II pre‐miRNAs, which have a 1‐nt 3′ overhang, TUT7 restores the canonical end structure (2‐nt 3′ overhang) through mono‐uridylation, thereby promoting miRNA biogenesis. For pre‐miRNAs where the 3′ end is further recessed into the stem (as in 3′ trimmed pre‐miRNAs), TUT7 generates an oligo‐U tail that leads to degradation. In contrast to Lin28‐stimulated oligo‐uridylation, which is processive, a distributive mode is employed by TUT7 for both mono‐ and oligo‐uridylation in the absence of Lin28. The overhang length dictates the frequency (but not duration) of the TUT7‐RNA interaction, thus explaining how TUT7 differentiates pre‐miRNA species with different overhangs. Our study reveals dual roles and mechanisms of uridylation in repair and removal of defective pre‐miRNAs.

RETRACTED: Cell Adhesion-Dependent Control of MicroRNA Decay

Mammalian microRNAs (miRNAs) are highly stable in most cell types, and their decay mechanism remains largely unknown. Here we report that some miRNAs degrade rapidly upon the loss of cell adhesion. When cells are grown at low density or cells are detached by trypsinization or EGTA treatment, mature miR-141 is downregulated while miR-200c from a common primary transcript (pri-miR-200c∼141) remains unaffected. Blockade of transcription by Actinomycin D leads to rapid depletion of miR-141 with a half-life of <1 hr when cells are detached, indicating that the regulation occurs via RNA decay. A sequence motif (UGUCU) in the center of miR-141 is necessary for the regulation. We further find that many other miRNAs including miR-200a, miR-34a, miR-29b, miR-301a, and miR-21 are degraded upon cell splitting. Induced destruction of persistent regulatory molecules such as miRNAs may increase cellular plasticity and facilitate cellular remodeling in response to the changes in cell adhesion.

Human UPF1 participates in small RNA-induced mRNA downregulation.

ABSTRACT MicroRNAs (miRNAs) are endogenous antisense regulators that trigger endonucleolytic mRNA cleavage, translational repression, and/or mRNA decay. miRNA-mediated gene regulation is important for numerous biological pathways, yet the underlying mechanisms are still under rigorous investigation. Here we identify human UPF1 (hUPF1) as a protein that contributes to RNA silencing. When hUPF1 is knocked down, miRNA targets are upregulated. The depletion of hUPF1 also increases the off-target messages of small interfering RNAs (siRNAs), which are imperfectly complementary to transfected siRNAs. Conversely, when overexpressed, wild-type hUPF1 downregulates miRNA targets. The helicase domain mutant of hUPF1 fails to suppress miRNA targets. hUPF1 interacts with human Argonaute 1 (hAGO1) and hAGO2 and colocalizes with hAGO1 and hAGO2 in processing bodies, which are known to be the sites for translational repression and mRNA destruction. We further find that the amounts of target messages bound to hAGO2 are reduced when hUPF1 is depleted. Our data thus suggest that hUPF1 may participate in RNA silencing by facilitating the binding of the RNA-induced silencing complex to the target and by accelerating the decay of the mRNA.

Mixed tailing by TENT4A and TENT4B shields mRNA from rapid deadenylation

A tale of not-so-pure tails The poly(A) tail of mRNA has been thought to be a pure stretch of adenosine nucleotides with little informational content except for length. Lim et al. identified enzymes that can decorate poly(A) tails with non-A nucleotides. The noncanonical poly(A) polymerases, TENT4A and TENT4B, incorporate intermittent non-A residues (G, U, or C) with a preference for guanosine, which results in a heterogenous poly(A) tail. Deadenylases trim poly(A) tails to initiate mRNA degradation but stall at the non-A residues. In effect, the not-so-pure tail stabilizes mRNAs by slowing down deadenylation. Science, this issue p. 701 The stabilizing role of mixed poly(A) tails in mRNA translation and decay is elucidated. RNA tails play integral roles in the regulation of messenger RNA (mRNA) translation and decay. Guanylation of the poly(A) tail was discovered recently, yet the enzymology and function remain obscure. Here we identify TENT4A (PAPD7) and TENT4B (PAPD5) as the enzymes responsible for mRNA guanylation. Purified TENT4 proteins generate a mixed poly(A) tail with intermittent non-adenosine residues, the most common of which is guanosine. A single guanosine residue is sufficient to impede the deadenylase CCR4-NOT complex, which trims the tail and exposes guanosine at the 3′ end. Consistently, depletion of TENT4A and TENT4B leads to a decrease in mRNA half-life and abundance in cells. Thus, TENT4A and TENT4B produce a mixed tail that shields mRNA from rapid deadenylation. Our study unveils the role of mixed tailing and expands the complexity of posttranscriptional gene regulation.