PNPT1 Release from Mitochondria during Apoptosis Triggers Decay of Poly(A) RNAs.

Widespread mRNA decay, an unappreciated feature of apoptosis, enhances cell death and depends on mitochondrial outer membrane permeabilization (MOMP), TUTases, and DIS3L2. Which RNAs are decayed and the decay-initiating event are unknown. Here, we show extensive decay of mRNAs and poly(A) noncoding (nc)RNAs at the 3' end, triggered by the mitochondrial intermembrane space 3'-to-5' exoribonuclease PNPT1, released during MOMP. PNPT1 knockdown inhibits apoptotic RNA decay and reduces apoptosis, while ectopic expression of PNPT1, but not an RNase-deficient mutant, increases RNA decay and cell death. The 3' end of PNPT1 substrates thread through a narrow channel. Many non-poly(A) ncRNAs contain 3'-secondary structures or bind proteins that may block PNPT1 activity. Indeed, mutations that disrupt the 3'-stem-loop of a decay-resistant ncRNA render the transcript susceptible, while adding a 3'-stem-loop to an mRNA prevents its decay. Thus, PNPT1 release from mitochondria during MOMP initiates apoptotic decay of RNAs lacking 3'-structures.

The RNase PARN Controls the Levels of Specific miRNAs that Contribute to p53 Regulation.

PARN loss-of-function mutations cause a severe form of the hereditary disease dyskeratosis congenita (DC). PARN deficiency affects the stability of non-coding RNAs such as human telomerase RNA (hTR), but these effects do not explain the severe disease in patients. We demonstrate that PARN deficiency affects the levels of numerous miRNAs in human cells. PARN regulates miRNA levels by stabilizing either mature or precursor miRNAs by removing oligo(A) tails added by the poly(A) polymerase PAPD5, which if remaining recruit the exonuclease DIS3L or DIS3L2 to degrade the miRNA. PARN knockdown destabilizes multiple miRNAs that repress p53 translation, which leads to an increase in p53 accumulation in a Dicer-dependent manner, thus explaining why PARN-defective patients show p53 accumulation. This work also reveals that DIS3L and DIS3L2 are critical 3' to 5' exonucleases that regulate miRNA stability, with the addition and removal of 3' end extensions controlling miRNA levels in the cell.

Overgrowth syndromes and pediatric cancers: how many roads lead to IGF2?

Overgrowth syndromes such as Perlman syndrome and associated pediatric cancers, including Wilms tumor, arise through genetic and, in certain instances, also epigenetic changes. In the case of the Beckwith-Wiedemann overgrowth syndrome and in Wilms tumor, increased levels of IGF2 have been shown to be causally related to the disease manifestation. In the previous issue of Genes & Development, Hunter and colleagues (pp. 903-908) investigated the molecular mechanisms by which mutations in the gene encoding the RNA degradation component DIS3L2 lead to Perlman syndrome. By analyzing nephron progenitor cells derived from their newly created Dis3l2 mutant mouse lines, the investigators showed that DIS3L2 loss of function leads to up-regulation of IGF2 independently of the let7 microRNA pathway. In a second study in this issue of Genes & Development, Chen and colleagues (pp. 996-1007) show that microRNA processing gene mutations in Wilms tumor lead to an increase in the levels of transcription factor pleomorphic adenoma gene 1 (PLAG1) that in turn activates IGF2 expression. Thus, augmented IGF2 expression seems to be a common downstream factor in both tissue overgrowth and Wilms tumor through several alternative mechanisms.

Loss of Dis3l2 partially phenocopies Perlman syndrome in mice and results in up-regulation of Igf2 in nephron progenitor cells.

Loss of function of the DIS3L2 exoribonuclease is associated with Wilms tumor and the Perlman congenital overgrowth syndrome. LIN28, a Wilms tumor oncoprotein, triggers the DIS3L2-mediated degradation of the precursor of let-7, a microRNA that inhibits Wilms tumor development. These observations have led to speculation that DIS3L2-mediated tumor suppression is attributable to let-7 regulation. Here we examine new DIS3L2-deficient cell lines and mouse models, demonstrating that DIS3L2 loss has no effect on mature let-7 levels. Rather, analysis of Dis3l2-null nephron progenitor cells, a potential cell of origin of Wilms tumors, reveals up-regulation of Igf2, a growth-promoting gene strongly associated with Wilms tumorigenesis. These findings nominate a new potential mechanism underlying the pathology associated with DIS3L2 deficiency.

DIS3L2 knockdown impairs key oncogenic properties of colorectal cancer cells via the mTOR signaling pathway.

DIS3L2 degrades different types of RNAs in an exosome-independent manner including mRNAs and several types of non-coding RNAs. DIS3L2-mediated degradation is preceded by the addition of nontemplated uridines at the 3'end of its targets by the terminal uridylyl transferases 4 and 7. Most of the literature that concerns DIS3L2 characterizes its involvement in several RNA degradation pathways, however, there is some evidence that its dysregulated activity may contribute to cancer development. In the present study, we characterize the role of DIS3L2 in human colorectal cancer (CRC). Using the public RNA datasets from The Cancer Genome Atlas (TCGA), we found higher DIS3L2 mRNA levels in CRC tissues versus normal colonic samples as well as worse prognosis in patients with high DIS3L2 expression. In addition, our RNA deep-sequencing data revealed that knockdown (KD) of DIS3L2 induces a strong transcriptomic disturbance in SW480 CRC cells. Moreover, gene ontology (GO) analysis of significant upregulated transcripts displays enrichment in mRNAs encoding proteins involved in cell cycle regulation and cancer-related pathways, which guided us to evaluate which specific hallmarks of cancer are differentially regulated by DIS3L2. To do so, we employed four CRC cell lines (HCT116, SW480, Caco-2 and HT-29) differing in their mutational background and oncogenicity. We demonstrate that depletion of DIS3L2 results in reduced cell viability of highly oncogenic SW480 and HCT116 CRC cells, but had little or no impact in the more differentiated Caco-2 and HT-29 cells. Remarkably, the mTOR signaling pathway, crucial for cell survival and growth, is downregulated after DIS3L2 KD, whereas AZGP1, an mTOR pathway inhibitor, is upregulated. Furthermore, our results indicate that depletion of DIS3L2 disturbs metastasis-associated properties, such as cell migration and invasion, only in highly oncogenic CRC cells. Our work reveals for the first time a role for DIS3L2 in sustaining CRC cell proliferation and provides evidence that this ribonuclease is required to support the viability and invasive behavior of dedifferentiated CRC cells.

Arabidopsis mRNA decay landscape arises from specialized RNA decay substrates, decapping-mediated feedback, and redundancy.

The decay of mRNA plays a vital role in modulating mRNA abundance, which, in turn, influences cellular and organismal processes. In plants and metazoans, three distinct pathways carry out the decay of most cytoplasmic mRNAs: The mRNA decapping complex, which requires the scaffold protein VARICOSE (VCS), removes a protective 5' cap, allowing for 5' to 3' decay via EXORIBONUCLEASE4 (XRN4, XRN1 in metazoans and yeast), and both the exosome and SUPPRESSOR OF VCS (SOV)/DIS3L2 degrade RNAs in the 3' to 5' direction. However, the unique biological contributions of these three pathways, and whether they degrade specialized sets of transcripts, are unknown. In Arabidopsis, the participation of SOV in RNA homeostasis is also unclear, because Arabidopsis sov mutants have a normal phenotype. We carried out mRNA decay analyses in wild-type, sov, vcs, and vcs sov seedlings, and used a mathematical modeling approach to determine decay rates and quantify gene-specific contributions of VCS and SOV to decay. This analysis revealed that VCS (decapping) contributes to decay of 68% of the transcriptome, and, while it initiates degradation of mRNAs with a wide range of decay rates, it especially contributes to decay of short-lived RNAs. Only a few RNAs were clear SOV substrates in that they decayed more slowly in sov mutants. However, 4,506 RNAs showed VCS-dependent feedback in sov that modulated decay rates, and, by inference, transcription, to maintain RNA abundances, suggesting that these RNAs might also be SOV substrates. This feedback was shown to be independent of siRNA activity.

TUT-DIS3L2 is a mammalian surveillance pathway for aberrant structured non-coding RNAs.

Uridylation of various cellular RNA species at the 3' end has been generally linked to RNA degradation. In mammals, uridylated pre-let-7 miRNAs and mRNAs are targeted by the 3' to 5' exoribonuclease DIS3L2. Mutations in DIS3L2 have been associated with Perlman syndrome and with Wilms tumor susceptibility. Using in vivo cross-linking and immunoprecipitation (CLIP) method, we discovered the DIS3L2-dependent cytoplasmic uridylome of human cells. We found a broad spectrum of uridylated RNAs including rRNAs, snRNAs, snoRNAs, tRNAs, vault, 7SL, Y RNAs, mRNAs, lncRNAs, and transcripts from pseudogenes. The unifying features of most of these identified RNAs are aberrant processing and the presence of stable secondary structures. Most importantly, we demonstrate that uridylation mediates DIS3L2 degradation of short RNA polymerase II-derived RNAs. Our findings establish the role of DIS3L2 and oligouridylation as the cytoplasmic quality control for highly structured ncRNAs.

Molecular basis for cytoplasmic RNA surveillance by uridylation-triggered decay in Drosophila.

The posttranscriptional addition of nucleotides to the 3' end of RNA regulates the maturation, function, and stability of RNA species in all domains of life. Here, we show that in flies, 3' terminal RNA uridylation triggers the processive, 3'-to-5' exoribonucleolytic decay via the RNase II/R enzyme CG16940, a homolog of the human Perlman syndrome exoribonuclease Dis3l2. Together with the TUTase Tailor, dmDis3l2 forms the cytoplasmic, terminal RNA uridylation-mediated processing (TRUMP) complex that functionally cooperates in the degradation of structured RNA RNA immunoprecipitation and high-throughput sequencing reveals a variety of TRUMP complex substrates, including abundant non-coding RNA, such as 5S rRNA, tRNA, snRNA, snoRNA, and the essential RNase MRP Based on genetic and biochemical evidence, we propose a key function of the TRUMP complex in the cytoplasmic quality control of RNA polymerase III transcripts. Together with high-throughput biochemical characterization of dmDis3l2 and bacterial RNase R, our results imply a conserved molecular function of RNase II/R enzymes as "readers" of destabilizing posttranscriptional marks-uridylation in eukaryotes and adenylation in prokaryotes-that play important roles in RNA surveillance.

Unraveling 3'-end RNA uridylation at nucleotide resolution.

Post-transcriptional modification of RNA, the so-called 'Epitranscriptome', can regulate RNA structure, stability, localization, and function. Numerous modifications have been identified in virtually all classes of RNAs, including messenger RNAs (mRNAs), transfer RNAs (tRNAs), ribosomal RNAs (rRNAs), microRNAs (miRNAs), and other noncoding RNAs (ncRNAs). These modifications may occur internally (by base or sugar modifications) and include RNA methylation at different nucleotide positions, or by the addition of various nucleotides at the 3'-end of certain transcripts by a family of terminal nucleotidylyl transferases. Developing methods to specifically and accurately detect and map these modifications is essential for understanding the molecular function(s) of individual RNA modifications and also for identifying and characterizing the proteins that may read, write, or erase them. Here, we focus on the characterization of RNA species targeted by 3' terminal uridylyl transferases (TUTases) (TUT4/7, also known as Zcchc11/6) and a 3'-5' exoribonuclease, Dis3l2, in the recently identified Dis3l2-mediated decay (DMD) pathway - a dedicated quality control pathway for a subset of ncRNAs. We describe the detailed methods used to precisely identify 3'-end modifications at nucleotide level resolution with a particular focus on the U1 and U2 small nuclear RNA (snRNA) components of the Spliceosome. These tools can be applied to investigate any RNA of interest and should facilitate studies aimed at elucidating the functional relevance of 3'-end modifications.

[Thyroid disruptor p, p'-DDE inhibited the expression of LHX4 and DIS3L protein in Nthy-ori-3-1 cells].

Objective: To observe the changes of LHX4 and DIS3L mRNA and protein expression in Nthy-ori-3-1 cells after the treatment of thyroid disruptor p, p'-DDE. Methods: Nthy-ori-3-1 cells in logarithmic growth phase were treated with 0, 0.5, 1.0, 2.0 and 5.0 µg/ml p, p'-DDE solution. The growth state and morphology of the cells were observed by microscope. The mRNA levels of LHX4 and DIS3L were detected by real-time fluorescent quantitative PCR, and the protein expression levels of LHX4 and DIS3L were detected by Western blot. Results: when the concentrations of p, p'-DDE were 0, 0.5, 1.0 and 2.0 µg/ml, Nthy-ori-3-1 cells grew normally. There were 33 differential genes in 2.0 µg/ml group, among which 13 genes were down regulated and 20 genes were up-regulated. Compared with the control group, the protein expression levels of LHX4 and DIS3L in 1.0 and 2.0 µg/ml groups were significantly decreased (P<0.05) , and the relative expression levels of LHX4 and DIS3L protein mRNA in 1.0 µg/ml group were significantly decreased (P<0.05) . Conclusion: p, p'-DDE can affect the protein expression of LHX4 and dis3l in nthy-ori-3-1 cells.

A role for DIS3L2 over natural nonsense-mediated mRNA decay targets in human cells.

The nonsense-mediated decay (NMD) pathway selectively degrades mRNAs carrying a premature translation-termination codon but also regulates the abundance of a large number of physiological mRNAs that encode full-length proteins. In human cells, NMD-targeted mRNAs are degraded by endonucleolytic cleavage and exonucleolytic degradation from both 5-' and 3'-ends. This is done by a process not yet completely understood that recruits decapping and 5'-to-3' exonuclease activities, as well as deadenylating and 3'-to-5' exonuclease exosome activities. In yeast, DIS3/Rrp44 protein is the catalytic subunit of the exosome, but in humans, there are three known paralogues of this enzyme: DIS3, DIS3L1, and DIS3L2. However, little is known about their role in NMD. Here, we show that some NMD-targets are DIS3L2 substrates in human cells. In addition, we observed that DIS3L2 acts over full-length transcripts, through a process that also involves UPF1. Moreover, DIS3L2-mediated decay is dependent on the activity of the terminal uridylyl transferases Zcchc6/11 (TUT7/4). Together, our findings establish a role for DIS3L2 and uridylation in NMD.

Regulation of RNA decay and cellular function by 3'-5' exoribonuclease DIS3L2.

DIS3L2, in which mutations have been linked to Perlman syndrome, is an RNA-binding protein with 3'-5' exoribonuclease activity. It contains two CSD domains and one S1 domain, all of which are RNA-binding domains, and one RNB domain that is responsible for the exoribonuclease activity. The 3' polyuridine of RNA substrates can serve as a degradation signal for DIS3L2. Because DIS3L2 is predominantly localized in the cytoplasm, it can recognize, bind, and mediate the degradation of cytoplasmic uridylated RNA, including pre-microRNA, mature microRNA, mRNA, and some other non-coding RNAs. Therefore, DIS3L2 plays an important role in cytoplasmic RNA surveillance and decay. DIS3L2 is involved in multiple biological and physiological processes such as cell division, proliferation, differentiation, and apoptosis. Nonetheless, the function of DIS3L2, especially its association with cancer, remains largely unknown. We summarize here the RNA substrates degraded by DIS3L2 with its exonucleolytic activity, together with the corresponding biological functions it is implicated in. Furthermore, we discuss whether DIS3L2 can function independently of its 3'-5' exoribonuclease activity, as well as its potential tumor-suppressive or oncogenic roles during cancer progression.

The Implication of mRNA Degradation Disorders on Human DISease: Focus on DIS3 and DIS3-Like Enzymes.

RNA degradation is considered a critical posttranscriptional regulatory checkpoint, maintaining the correct functioning of organisms. When a specific RNA transcript is no longer required in the cell, it is signaled for degradation through a number of highly regulated steps. Ribonucleases (or simply RNases) are key enzymes involved in the control of RNA stability. These enzymes can perform the RNA degradation alone or cooperate with other proteins in RNA degradation complexes. Important findings over the last years have shed light into eukaryotic RNA degradation by members of the RNase II/RNB family of enzymes. DIS3 enzyme belongs to this family and represents one of the catalytic subunits of the multiprotein complex exosome. This RNase has a diverse range of functions, mainly within nuclear RNA metabolism. Humans encode two other DIS3-like enzymes: DIS3L (DIS3L1) and DIS3L2. DIS3L1 also acts in association with the exosome but is strictly cytoplasmic. In contrast, DIS3L2 acts independently of the exosome and shows a distinctive preference for uridylated RNAs. These enzymes have been shown to be involved in important cellular processes, such as mitotic control, and associated with human disorders like cancer. This review shows how the impairment of function of each of these enzymes is implicated in human disease.

Long term survival of a patient with Perlman syndrome due to novel compound heterozygous missense mutations in RNB domain of DIS3L2.

Perlman syndrome is a rare overgrowth syndrome characterized by polyhydramnios, macrosomia, distinctive facial appearance, renal dysplasia, and a predisposition to Wilms' tumor. The syndrome is often associated with a high neonatal mortality rate and there are few reports of long-term survivors. We studied a 6-year-old Japanese female patient, who was diagnosed with Perlman syndrome, with novel compound heterozygous mutations in DIS3L2 (c.[367-2A > G];[1328T > A]), who has survived long term. Most reported DIS3L2 mutations have been the homozygous deletion of exon 6 or exon 9, and these mutations would certainly have caused the loss of both RNA binding and degradation activity. We have identified new compound heterozygous mutations in the DIS3L2 of this long-term survivor of Perlman syndrome. The reason our patient has survived long-term would be a missense mutation (c.1328 T > A, p.Met443Lys) having retained RNA binding in both the cold-shock domains and the S1 domain, and through partial RNA degradation. If partial exonuclease functions remain in at least one allele, long-term survival may be possible. Further studies of Perlman syndrome patients with proven DIS3L2 mutations are needed to clarify genotype-phenotype correlation.

The roles of the exoribonucleases DIS3L2 and XRN1 in human disease.

RNA degradation is a vital post-transcriptional process which ensures that transcripts are maintained at the correct level within the cell. DIS3L2 and XRN1 are conserved exoribonucleases that are critical for the degradation of cytoplasmic RNAs. Although the molecular mechanisms of RNA degradation by DIS3L2 and XRN1 have been well studied, less is known about their specific roles in the development of multicellular organisms or human disease. This review focusses on the roles of DIS3L2 and XRN1 in the pathogenesis of human disease, particularly in relation to phenotypes seen in model organisms. The known diseases associated with loss of activity of DIS3L2 and XRN1 are discussed, together with possible mechanisms and cellular pathways leading to these disease conditions.

Structural analysis of Dis3l2, an exosome-independent exonuclease from Schizosaccharomyces pombe.

After deadenylation and decapping, cytoplasmic mRNA can be digested in two opposite directions: in the 5'-3' direction by Xrn1 or in the 3'-5' direction by the exosome complex. Recently, a novel 3'-5' RNA-decay pathway involving Dis3l2 has been described that differs from degradation by Xrn1 and the exosome. The product of the Schizosaccharomyces pombe gene SPAC2C4.07c was identified as a homologue of human Dis3l2. In this work, the 2.8 Å resolution X-ray crystal structure of S. pombe Dis3l2 (SpDis3l2) is reported, the conformation of which is obviously different from that in the homologous mouse Dis3l2-RNA complex. Fluorescence polarization assay experiments showed that RNB and S1 are the primary RNA-binding domains and that the CSDs (CSD1 and CSD2) play an indispensable role in the RNA-binding process of SpDis3l2. Taking the structure comparison and mutagenic experiments together, it can be inferred that the RNA-recognition pattern of SpDis3l2 resembles that of its mouse homologue rather than that of the Escherichia coli RNase II-RNA complex. Furthermore, a drastic conformation change could occur following the binding of the RNA substrate to SpDis3l2.

A role of uridylation pathway for blockade of let-7 microRNA biogenesis by Lin28B.

The precise control of microRNA (miRNA) biosynthesis is crucial for gene regulation. Lin28A and Lin28B are selective inhibitors of biogenesis of let-7 miRNAs involved in development and tumorigenesis. Lin28A selectively inhibits let-7 biogenesis through cytoplasmic uridylation of precursor let-7 by TUT4 terminal uridyl transferase and subsequent degradation by Dis3l2 exonuclease. However, a role of this uridylation pathway remains unclear in let-7 blockade by Lin28B, a paralog of Lin28A, while Lin28B is reported to engage a distinct mechanism in the nucleus to suppress let-7. Here we revisit a functional link between Lin28B and the uridylation pathway with a focus on let-7 metabolism in cancer cells. Both Lin28A and Lin28B interacted with Dis3l2 in the cytoplasm, and silencing of Dis3l2 upregulated uridylated pre-let-7 in both Lin28A- and Lin28B-expressing cancer cell lines. In addition, we found that amounts of let-7 precursors influenced intracellular localization of Lin28B. Furthermore, we found that MCPIP1 (Zc3h12a) ribonuclease was also involved in degradation of both non-uridylated and uridylated pre-let-7. Cancer transcriptome analysis showed association of expression levels of Lin28B and uridylation pathway components, TUT4 and Dis3l2, in various human cancer cells and hepatocellular carcinoma. Collectively, these results suggest that cytoplasmic uridylation pathway actively participates in blockade of let-7 biogenesis by Lin28B.

Mammalian DIS3L2 exoribonuclease targets the uridylated precursors of let-7 miRNAs.

The mechanisms of gene expression regulation by miRNAs have been extensively studied. However, the regulation of miRNA function and decay has long remained enigmatic. Only recently, 3' uridylation via LIN28A-TUT4/7 has been recognized as an essential component controlling the biogenesis of let-7 miRNAs in stem cells. Although uridylation has been generally implicated in miRNA degradation, the nuclease responsible has remained unknown. Here, we identify the Perlman syndrome-associated protein DIS3L2 as an oligo(U)-binding and processing exoribonuclease that specifically targets uridylated pre-let-7 in vivo. This study establishes DIS3L2 as the missing component of the LIN28-TUT4/7-DIS3L2 pathway required for the repression of let-7 in pluripotent cells.

Loss of DIS3L in the initial segment is dispensable for sperm maturation in the epididymis and male fertility.

DIS3L, a catalytic exoribonuclease associated with the cytoplasmic exosome complex, degrades cytoplasmic RNAs and is implicated in cancers and certain other diseases in humans. Epididymis plays a pivotal role in the transport, maturation, and storage of sperm required for male fertility. However, it remains unclear whether DIS3L-mediated cytoplasmic RNA degradation plays a role in epididymis biology and functioning. Herein, we fabricated a Dis3l conditional knockout (Dis3l cKO) mouse line in which DIS3L was ablated from the principal cells of the initial segment (IS). Morphological analyses showed that spermatogenesis and IS differentiation occurred normally in Dis3l cKO mice. Additionally, the absence of DIS3L had no dramatic influence on the transcriptome of IS. Moreover, the sperm count, morphology, motility, and acrosome reaction frequency in Dis3l cKO mice were comparable to that of the control, indicating that the Dis3l cKO males had normal fertility. Collectively, our genetic model demonstrates that DIS3L inactivation in the IS is nonessential for sperm maturation and male fertility.

Characterisation of the in-vivo miRNA landscape in Drosophila ribonuclease mutants reveals Pacman-mediated regulation of the highly conserved let-7 cluster during apoptotic processes.

The control of gene expression is a fundamental process essential for correct development and to maintain homeostasis. Many post-transcriptional mechanisms exist to maintain the correct levels of each RNA transcript within the cell. Controlled and targeted cytoplasmic RNA degradation is one such mechanism with the 5'-3' exoribonuclease Pacman (XRN1) and the 3'-5' exoribonuclease Dis3L2 playing crucial roles. Loss of function mutations in either Pacman or Dis3L2 have been demonstrated to result in distinct phenotypes, and both have been implicated in human disease. One mechanism by which gene expression is controlled is through the function of miRNAs which have been shown to be crucial for the control of almost all cellular processes. Although the biogenesis and mechanisms of action of miRNAs have been comprehensively studied, the mechanisms regulating their own turnover are not well understood. Here we characterise the miRNA landscape in a natural developing tissue, the Drosophila melanogaster wing imaginal disc, and assess the importance of Pacman and Dis3L2 on the abundance of miRNAs. We reveal a complex landscape of miRNA expression and show that whilst a null mutation in dis3L2 has a minimal effect on the miRNA expression profile, loss of Pacman has a profound effect with a third of all detected miRNAs demonstrating Pacman sensitivity. We also reveal a role for Pacman in regulating the highly conserved let-7 cluster (containing miR-100, let-7 and miR-125) and present a genetic model outlining a positive feedback loop regulated by Pacman which enhances our understanding of the apoptotic phenotype observed in Pacman mutants.