Mixed tailing by TENT4A and TENT4B shields mRNA from rapid deadenylation.

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.

PABP Cooperates with the CCR4-NOT Complex to Promote mRNA Deadenylation and Block Precocious Decay.

Multiple deadenylases are known in vertebrates, the PAN2-PAN3 (PAN2/3) and CCR4-NOT (CNOT) complexes, and PARN, yet their differential functions remain ambiguous. Moreover, the role of poly(A) binding protein (PABP) is obscure, limiting our understanding of the deadenylation mechanism. Here, we show that CNOT serves as a predominant nonspecific deadenylase for cytoplasmic poly(A)+ RNAs, and PABP promotes deadenylation while preventing premature uridylation and decay. PAN2/3 selectively trims long tails (>∼150 nt) with minimal effect on transcriptome, whereas PARN does not affect mRNA deadenylation. CAF1 and CCR4, catalytic subunits of CNOT, display distinct activities: CAF1 trims naked poly(A) segments and is blocked by PABPC, whereas CCR4 is activated by PABPC to shorten PABPC-protected sequences. Concerted actions of CAF1 and CCR4 delineate the ∼27 nt periodic PABPC footprints along shortening tail. Our study unveils distinct functions of deadenylases and PABPC, re-drawing the view on mRNA deadenylation and regulation.

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.

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.

Regulation of microRNA biogenesis.

MicroRNAs (miRNAs) are small non-coding RNAs that function as guide molecules in RNA silencing. Targeting most protein-coding transcripts, miRNAs are involved in nearly all developmental and pathological processes in animals. The biogenesis of miRNAs is under tight temporal and spatial control, and their dysregulation is associated with many human diseases, particularly cancer. In animals, miRNAs are ∼22 nucleotides in length, and they are produced by two RNase III proteins--Drosha and Dicer. miRNA biogenesis is regulated at multiple levels, including at the level of miRNA transcription; its processing by Drosha and Dicer in the nucleus and cytoplasm, respectively; its modification by RNA editing, RNA methylation, uridylation and adenylation; Argonaute loading; and RNA decay. Non-canonical pathways for miRNA biogenesis, including those that are independent of Drosha or Dicer, are also emerging.

TAIL-seq: genome-wide determination of poly(A) tail length and 3' end modifications.

Global investigation of the 3' extremity of mRNA (3'-terminome), despite its importance in gene regulation, has not been feasible due to technical challenges associated with homopolymeric sequences and relative paucity of mRNA. We here develop a method, TAIL-seq, to sequence the very end of mRNA molecules. TAIL-seq allows us to measure poly(A) tail length at the genomic scale. Median poly(A) length is 50-100 nt in HeLa and NIH 3T3 cells. Poly(A) length correlates with mRNA half-life, but not with translational efficiency. Surprisingly, we discover widespread uridylation and guanylation at the downstream of poly(A) tail. The U tails are generally attached to short poly(A) tails (<25 nt), while the G tails are found mainly on longer poly(A) tails (>40 nt), implicating their generic roles in mRNA stability control. TAIL-seq is a potent tool to dissect dynamic control of mRNA turnover and translational control, and to discover unforeseen features of RNA cleavage and tailing.

LIN28A is a suppressor of ER-associated translation in embryonic stem cells.

LIN28 plays a critical role in developmental transition, glucose metabolism, and tumorigenesis. At the molecular level, LIN28 is known to repress maturation of let-7 microRNAs and enhance translation of certain mRNAs. In this study, we obtain a genome-wide view of the molecular function of LIN28A in mouse embryonic stem cells by carrying out RNA crosslinking-immunoprecipitation-sequencing (CLIP-seq) and ribosome footprinting. We find that, in addition to let-7 precursors, LIN28A binds to a large number of spliced mRNAs. LIN28A recognizes AAGNNG, AAGNG, and less frequently UGUG, which are located in the terminal loop of a small hairpin. LIN28A is localized to the periendoplasmic reticulum (ER) area and inhibits translation of mRNAs that are destined for the ER, reducing the synthesis of transmembrane proteins, ER or Golgi lumen proteins, and secretory proteins. Our study suggests a selective regulatory mechanism for ER-associated translation and reveals an unexpected role of LIN28A as a global suppressor of genes in the secretory pathway.

Mono-uridylation of pre-microRNA as a key step in the biogenesis of group II let-7 microRNAs.

RNase III Drosha initiates microRNA (miRNA) maturation by cleaving a primary miRNA transcript and releasing a pre-miRNA with a 2 nt 3' overhang. Dicer recognizes the 2 nt 3' overhang structure to selectively process pre-miRNAs. Here, we find that, unlike prototypic pre-miRNAs (group I), group II pre-miRNAs acquire a shorter (1 nt) 3' overhang from Drosha processing and therefore require a 3'-end mono-uridylation for Dicer processing. The majority of let-7 and miR-105 belong to group II. We identify TUT7/ZCCHC6, TUT4/ZCCHC11, and TUT2/PAPD4/GLD2 as the terminal uridylyl transferases responsible for pre-miRNA mono-uridylation. The TUTs act specifically on dsRNAs with a 1 nt 3' overhang, thereby creating a 2 nt 3' overhang. Depletion of TUTs reduces let-7 levels and disrupts let-7 function. Although the let-7 suppressor, Lin28, induces inhibitory oligo-uridylation in embryonic stem cells, mono-uridylation occurs in somatic cells lacking Lin28 to promote let-7 biogenesis. Our study reveals functional duality of uridylation and introduces TUT7/4/2 as components of the miRNA biogenesis pathway.

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.

Inhibition of prostate cancer using RNA interference-directed knockdown of platelet-derived growth factor receptor.

OBJECTIVES: To determine whether platelet-derived growth factor receptor (PDGFR) plays a role in the tumorigenicity of prostate cancer cells. METHODS: PC3 prostate cancer cells were transfected with small interfering (si)PDGFR-α and siPDGFR-ß, constructed according to the conventional small interfering RNA design standard. Reverse transcriptase polymerase chain reaction, Western blot analysis, and cell growth were studied to determine the characteristics of PDGFR-α and PDGFR-ß in vitro. The prostate cancer xenograft model was established to investigate whether knockout of PDGFR-α and PDGFR-ß decreases prostate cancer tumor growth in vivo. The experimental groups were defined as group 1 (PC3 cells only), group 2 (PC3 cells transfected with small interfering green fluorescent protein), group 3 (PC3 cells transfected with siPDGFR-α), group 4 (PC3 cells transfected with siPDGFR-ß), and group 5 (PC3 cells transfected with siPDGFR-α and siPDGFR-ß). RESULTS: Western blot analysis revealed that siPDGFR-α and siPDGFR-ß significantly blocked PDGFR-α and PDGFR-ß protein expression. After 48 hours of transfection of the PC3 cells with siPDGFR-α and siPDGFR-ß, the relative fractions of viable cells were reduced to 47.7% (P = .007) and 38.5% (P = .010). In vivo, mice treated with siPDGFR-α or siPDGFR-ß and siPDGFR-α plus siPDGFR-ß had significant tumor cell growth arrest compared with the mice in groups 1 and 2 (P = .001). In addition, a significant reduction in the microvessel density was observed in tumors from the mice treated with siPDGFR-α or siPDGFR-ß and siPDGFR-α plus siPDGFR-ß (P < .001). CONCLUSIONS: The results of the present study suggest that siPDGFR-α and siPDGFR-ß might inhibit prostate cancer cell growth by the suppression of angiogenesis.

Human UPF1 participates in small RNA-induced mRNA downregulation.

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.

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.

miR-29 miRNAs activate p53 by targeting p85 alpha and CDC42.

The tumor suppressor p53 is central to many cellular stress responses. Although numerous protein factors that control p53 have been identified, the role of microRNAs (miRNAs) in regulating p53 remains unexplored. In a screen for miRNAs that modulate p53 activity, we find that miR-29 family members (miR-29a, miR-29b and miR-29c) upregulate p53 levels and induce apoptosis in a p53-dependent manner. We further find that miR-29 family members directly suppress p85 alpha (the regulatory subunit of PI3 kinase) and CDC42 (a Rho family GTPase), both of which negatively regulate p53. Our findings provide new insights into the role of miRNAs in the p53 pathway.

Lin28 mediates the terminal uridylation of let-7 precursor MicroRNA.

The precise control of microRNA (miRNA) biogenesis is critical for embryonic development and normal cellular functions, and its dysregulation is often associated with human diseases. Though the birth and maturation pathway of miRNA has been established, the regulation and death pathway remains largely unknown. Here, we report the RNA-binding proteins, Lin28a and Lin28b, as posttranscriptional repressors of let-7 miRNA biogenesis. We observe that the Lin28 proteins act mainly in the cytoplasm by inducing uridylation of precursor let-7 (pre-let-7) at its 3' end. The uridylated pre-let-7 (up-let-7) fails Dicer processing and undergoes degradation. We provide a mechanism for the posttranscriptional regulation of miRNA biogenesis by Lin28 which is highly expressed in undifferentiated cells and certain cancer cells. The Lin28-mediated downregulation of let-7 may play a key role in development, stem cell programming, and tumorigenesis.