LIN28B alters ribosomal dynamics to promote metastasis in MYCN-driven malignancy.

High expression of LIN28B is associated with aggressive malignancy and poor survival. Here, probing MYCN-amplified neuroblastoma as a model system, we showed that LIN28B expression was associated with enhanced cell migration in vitro and invasive and metastatic behavior in murine xenografts. Sequence analysis of the polyribosome fraction of LIN28B-expressing neuroblastoma cells revealed let-7-independent enrichment of transcripts encoding components of the translational and ribosomal apparatus and depletion of transcripts of neuronal developmental programs. We further observed that LIN28B utilizes both its cold shock and zinc finger RNA binding domains to preferentially interact with MYCN-induced transcripts of the ribosomal complex, enhancing their translation. These data demonstrated that LIN28B couples the MYCN-driven transcriptional program to enhanced ribosomal translation, thereby implicating LIN28B as a posttranscriptional driver of the metastatic phenotype.

METTL1-mediated m7G modification of Arg-TCT tRNA drives oncogenic transformation.

The emerging "epitranscriptomics" field is providing insights into the biological and pathological roles of different RNA modifications. The RNA methyltransferase METTL1 catalyzes N7-methylguanosine (m7G) modification of tRNAs. Here we find METTL1 is frequently amplified and overexpressed in cancers and is associated with poor patient survival. METTL1 depletion causes decreased abundance of m7G-modified tRNAs and altered cell cycle and inhibits oncogenicity. Conversely, METTL1 overexpression induces oncogenic cell transformation and cancer. Mechanistically, we find increased abundance of m7G-modified tRNAs, in particular Arg-TCT-4-1, and increased translation of mRNAs, including cell cycle regulators that are enriched in the corresponding AGA codon. Accordingly, Arg-TCT expression is elevated in many tumor types and is associated with patient survival, and strikingly, overexpression of this individual tRNA induces oncogenic transformation. Thus, METTL1-mediated tRNA modification drives oncogenic transformation through a remodeling of the mRNA "translatome" to increase expression of growth-promoting proteins and represents a promising anti-cancer target.

Global miRNA dosage control of embryonic germ layer specification.

MicroRNAs (miRNAs) have essential functions during embryonic development, and their dysregulation causes cancer1,2. Altered global miRNA abundance is found in different tissues and tumours, which implies that precise control of miRNA dosage is important1,3,4, but the underlying mechanism(s) of this control remain unknown. The protein complex Microprocessor, which comprises one DROSHA and two DGCR8 proteins, is essential for miRNA biogenesis5-7. Here we identify a developmentally regulated miRNA dosage control mechanism that involves alternative transcription initiation (ATI) of DGCR8. ATI occurs downstream of a stem-loop in DGCR8 mRNA to bypass an autoregulatory feedback loop during mouse embryonic stem (mES) cell differentiation. Deletion of the stem-loop causes imbalanced DGCR8:DROSHA protein stoichiometry that drives irreversible Microprocessor aggregation, reduced primary miRNA processing, decreased mature miRNA abundance, and widespread de-repression of lipid metabolic mRNA targets. Although global miRNA dosage control is not essential for mES cells to exit from pluripotency, its dysregulation alters lipid metabolic pathways and interferes with embryonic development by disrupting germ layer specification in vitro and in vivo. This miRNA dosage control mechanism is conserved in humans. Our results identify a promoter switch that balances Microprocessor autoregulation and aggregation to precisely control global miRNA dosage and govern stem cell fate decisions during early embryonic development.

Calmodulin inhibitors improve erythropoiesis in Diamond-Blackfan anemia.

Diamond-Blackfan anemia (DBA) is a rare hematopoietic disease characterized by a block in red cell differentiation. Most DBA cases are caused by mutations in ribosomal proteins and characterized by higher than normal activity of the tumor suppressor p53. Higher p53 activity is thought to contribute to DBA phenotypes by inducing apoptosis during red blood cell differentiation. Currently, there are few therapies available for patients with DBA. We performed a chemical screen using zebrafish ribosomal small subunit protein 29 (rps29) mutant embryos that have a p53-dependent anemia and identified calmodulin inhibitors that rescued the phenotype. Our studies demonstrated that calmodulin inhibitors attenuated p53 protein amount and activity. Treatment with calmodulin inhibitors led to decreased p53 translation and accumulation but does not affect p53 stability. A U.S. Food and Drug Administration-approved calmodulin inhibitor, trifluoperazine, rescued hematopoietic phenotypes of DBA models in vivo in zebrafish and mouse models. In addition, trifluoperazine rescued these phenotypes in human CD34+ hematopoietic stem and progenitor cells. Erythroid differentiation was also improved in CD34+ cells isolated from a patient with DBA. This work uncovers a potential avenue of therapeutic development for patients with DBA.

The Perlman syndrome DIS3L2 exoribonuclease safeguards endoplasmic reticulum-targeted mRNA translation and calcium ion homeostasis.

DIS3L2-mediated decay (DMD) is a surveillance pathway for certain non-coding RNAs (ncRNAs) including ribosomal RNAs (rRNAs), transfer RNAs (tRNAs), small nuclear RNAs (snRNAs), and RMRP. While mutations in DIS3L2 are associated with Perlman syndrome, the biological significance of impaired DMD is obscure and pathological RNAs have not been identified. Here, by ribosome profiling (Ribo-seq) we find specific dysregulation of endoplasmic reticulum (ER)-targeted mRNA translation in DIS3L2-deficient cells. Mechanistically, DMD functions in the quality control of the 7SL ncRNA component of the signal recognition particle (SRP) required for ER-targeted translation. Upon DIS3L2 loss, sustained 3'-end uridylation of aberrant 7SL RNA impacts ER-targeted translation and causes ER calcium leakage. Consequently, elevated intracellular calcium in DIS3L2-deficient cells activates calcium signaling response genes and perturbs ESC differentiation. Thus, DMD is required to safeguard ER-targeted mRNA translation, intracellular calcium homeostasis, and stem cell differentiation.

Exonuclease requirements for mammalian ribosomal RNA biogenesis and surveillance.

Ribosomal RNA (rRNA) biogenesis is a multistep process requiring several nuclear and cytoplasmic exonucleases. The exact processing steps for mammalian 5.8S rRNA remain obscure. Here, using loss-of-function approaches in mouse embryonic stem cells (mESCs) and deep sequencing of rRNA intermediates, we investigate the requirements of exonucleases known to be involved in 5.8S maturation at nucleotide resolution and explore the role of the Perlman syndrome-associated 3'-5' exonuclease Dis3l2 in rRNA processing. We uncover a novel cytoplasmic intermediate that we name '7SB' rRNA that is generated through sequential processing by distinct exosome complexes. 7SB rRNA can be oligoadenylated by an unknown enzyme and/or oligouridylated by TUT4/7 and subsequently processed by Dis3l2 and Eri1. Moreover, exosome depletion triggers Dis3l2-mediated decay (DMD) as a surveillance pathway for rRNAs. Our data identify previously unknown 5.8S rRNA processing steps and provide nucleotide-level insight into the exonuclease requirements for mammalian rRNA processing.

Dis3l2-Mediated Decay Is a Quality Control Pathway for Noncoding RNAs.

Mutations in the 3'-5' exonuclease DIS3L2 are associated with Perlman syndrome and hypersusceptibility to Wilms tumorigenesis. Previously, we found that Dis3l2 specifically recognizes and degrades uridylated pre-let-7 microRNA. However, the widespread relevance of Dis3l2-mediated decay of uridylated substrates remains unknown. Here, we applied an unbiased RNA immunoprecipitation strategy to identify Dis3l2 targets in mouse embryonic stem cells. The disease-associated long noncoding RNA (lncRNA) Rmrp, 7SL, as well as several other Pol III-transcribed noncoding RNAs (ncRNAs) were among the most highly enriched Dis3l2-bound RNAs. 3'-Uridylated Rmrp, 7SL, and small nuclear RNA (snRNA) species were highly stabilized in the cytoplasm of Dis3l2-depleted cells. Deep sequencing analysis of Rmrp 3' ends revealed extensive oligouridylation mainly on transcripts with imprecise ends. We implicate the terminal uridylyl transferases (TUTases) Zcchc6/11 in the uridylation of these ncRNAs, and biochemical reconstitution assays demonstrate the sufficiency of TUTase-Dis3l2 for Rmrp decay. This establishes Dis3l2-mediated decay (DMD) as a quality-control pathway that eliminates aberrant ncRNAs.

A Single Let-7 MicroRNA Bypasses LIN28-Mediated Repression.

Let-7 microRNAs (miRNAs) are critical regulators of animal development, stem cell differentiation, glucose metabolism, and tumorigenesis. Mammalian genomes contain 12 let-7 isoforms that suppress expression of a common set of target mRNAs. LIN28 proteins selectively block let-7 biogenesis in undifferentiated cells and in cancer. The current model for coordinate let-7 repression involves the LIN28 cold-shock domain (CSD) binding the terminal loop and the two CCHC-type zinc fingers recognizing a GGAG sequence motif in precursor let-7 (pre-let-7) RNAs. Here, we perform a systematic analysis of all let-7 miRNAs and find that a single let-7 family member, human let-7a-3 (and its murine ortholog let-7c-2), escapes LIN28-mediated regulation. Mechanistically, we find that the pre-let-7c-2 loop precludes LIN28A binding and regulation. These findings refine the current model of let-7 regulation by LIN28 proteins and have important implications for understanding the LIN28/let-7 axis in development and disease.

Destabilization of pluripotency in the absence of Mad2l2.

The induction and maintenance of pluripotency requires the expression of several core factors at appropriate levels (Oct4, Sox2, Klf4, Prdm14). A subset of these proteins (Oct4, Sox2, Prdm14) also plays crucial roles for the establishment of primordial germ cells (PGCs). Here we demonstrate that the Mad2l2 (MAD2B, Rev7) gene product is not only required by PGCs, but also by pluripotent embryonic stem cells (ESCs), depending on the growth conditions. Mad2l2(-/-) ESCs were unstable in LIF/serum medium, and differentiated into primitive endoderm. However, they could be stably propagated using small molecule inhibitors of MAPK signaling. Several components of the MAPK cascade were up- or downregulated even in undifferentiated Mad2l2(-/-) ESCs. Global levels of repressive histone H3 variants were increased in mutant ESCs, and the epigenetic signatures on pluripotency-, primitive endoderm-, and MAPK-related loci differed. Thus, H3K9me2 repressed the Nanog promoter, while the promoter of Gata4 lost H3K27me3 and became de-repressed in LIF/serum condition. Promoters associated with genes involved in MAPK signaling also showed misregulation of these histone marks. Such epigenetic modifications could be indirect consequences of mutating Mad2l2. However, our previous observations suggested the histone methyltransferases as direct (G9a) or indirect (Ezh2) targets of Mad2l2. In effect, the intricate balance necessary for pluripotency becomes perturbed in the absence of Mad2l2.

Behavior of mouse spermatogonial stem-like cells on an electrospun nanofibrillar matrix.

PURPOSE: Spermatogonial stem cells are affected by the interactions of extrinsic signals produced by components of the microenvironment niche, in addition to the chemical and physical properties of the extracellular matrix. Therefore, this study was initiated to assess the interaction of these cells on a synthetic nanofibrillar extracellular matrix that mimicked the geometry and nanotopography of the basement membrane for cellular growth. METHODS: This study has used a variety of experimental approaches to investigate the interaction of mouse neonatal-derived spermatogonial stem-like cells on a synthetic random oriented three-dimensional nanofibrillar matrix composed of electrospun polyamide nanofibers (Ultra-Web™). RESULTS: Spermatogonial stem-like cell colonies were characterized by their ability to express α6-integrin, Thy-1, PLZF, and ß1-integrin. After culture of cells on the nanofibrillar surfaces for 7 days, the number of colonies, the number of cells in each colony, and the average area of colonies were increased (P < 0.05). However, the expression difference of related markers in both groups was not significant. A significantly higher proliferation and survival was observed in the nanofibrillar group (P < 0.05). After transplantation into the testes of busulfan-treated adult mice, spermatogonial stem-like cell colonies that were cultured on the nanofibrillar surface demonstrated functionality, as verified by their ability to migrate to the seminiferous basal membrane, where they produced additional colonies. CONCLUSIONS: These results have suggested that electrospun nanofibrillar surfaces could provide a more favorable microenvironment for in vitro short term culture of spermatogonial stem-like cell colonies.

Comparison of Neonatal and Adult Mice-derived Sertoli Cells in Support of Expansion of Mouse Spermatogonial Stem Cells In vitro.

BACKGROUND: This study compared neonatal and adult mice-derived Sertoli cells (NSCs and ASCs) to examine the influence of feeder cells derived from donors of different ages on the maintenance of mouse spermatogonial stem cells (SSCs) in vitro. MATERIALS AND METHODS: SSCs were derived from the testes of six-day-old mice. They were subsequently transferred to Sertoli cells which were isolated by datura stramonium agglutinin (DSA) lectin from neonatal and adult mice for five days. RESULTS: The numbers of spermatogonial colonies, the numbers of cells per colony, and cloning efficiency were assessed in presence of NSCs and ASCs. The expression of α6- and ß1-integrin- positive cells was evaluated. Moreover, the functionality of the cells was assessed by their transplantation into the testes of busulfan-induced infertile mice. Colony efficiency assay showed that the number of colonies derived from single spermatogonial cells were significantly higher on NSCs. Additionally, the transplantation of dissociated colonies into the testes of busulfan-induced infertile mice showed their migration to the seminiferous basal membrane. CONCLUSION: These results show that NSCs may provide a more favorable microenvironment in comparison with ASCs for in vitro culture of spermatogonial colonies.