Loss of MBNL leads to disruption of developmentally regulated alternative polyadenylation in RNA-mediated disease.

Inhibition of muscleblind-like (MBNL) activity due to sequestration by microsatellite expansion RNAs is a major pathogenic event in the RNA-mediated disease myotonic dystrophy (DM). Although MBNL1 and MBNL2 bind to nascent transcripts to regulate alternative splicing during muscle and brain development, another major binding site for the MBNL protein family is the 3' untranslated region of target RNAs. Here, we report that depletion of Mbnl proteins in mouse embryo fibroblasts leads to misregulation of thousands of alternative polyadenylation events. HITS-CLIP and minigene reporter analyses indicate that these polyadenylation switches are a direct consequence of MBNL binding to target RNAs. Misregulated alternative polyadenylation also occurs in skeletal muscle in a mouse polyCUG model and human DM, resulting in the persistence of neonatal polyadenylation patterns. These findings reveal an additional developmental function for MBNL proteins and demonstrate that DM is characterized by misregulation of pre-mRNA processing at multiple levels.

Global insights into alternative polyadenylation regulation.

Alternative pre-mRNA processing greatly increases the coding capacity of the human genome and regulatory factors involved in RNA processing play critical roles in tissue development and maintenance. Indeed, abnormal functions of RNA processing factors have been associated with a wide range of human diseases from cancer to neurodegenerative disorders. While many studies have emphasized the importance of alternative splicing (AS), recent high-throughput sequencing efforts have also allowed global surveys of alternative polyadenylation (APA). For the majority of pre-mRNAs, as well as some non-coding transcripts such as lncRNAs, APA selects different 3'-ends and thus modulates the availability of regulatory sites recognized by trans-acting regulatory effectors, including miRs and RNA binding proteins (RBPs). Here, we compare the available technologies for assessing global polyadenylation patterns, summarize the roles of auxiliary factors on APA, and discuss the impact of differential polyA site (pA) selection in the determination of cell fate, transformation and disease.

Converging evidence of mitochondrial dysfunction in a yeast model of homocysteine metabolism imbalance.

An elevated level of homocysteine, a thiol amino acid, is associated with various complex disorders. The cellular effects of homocysteine and its precursors S-adenosylhomocysteine (AdoHcy) and S-adenosylmethionine (AdoMet) are, however, poorly understood. We used Saccharomyces cerevisiae as a model to understand the basis of pathogenicity induced by homocysteine and its precursors. Both homocysteine and AdoHcy but not AdoMet inhibited the growth of the str4Δ strain (which lacks the enzyme that converts homocysteine to cystathionine-mimicking vascular cells). Addition of AdoMet abrogated the inhibitory effect of AdoHcy but not that of homocysteine indicating that an increase in the AdoMet/AdoHcy ratio is sufficient to overcome the AdoHcy-mediated growth defect but not that of homocysteine. Also, the transcriptomic profile of AdoHcy and homocysteine showed gross dissimilarity based on gene enrichment analysis. Furthermore, compared with homocysteine, AdoHcy treatment caused a higher level of oxidative stress in the cells. However, unlike a previously reported response in wild type (Kumar, A., John, L., Alam, M. M., Gupta, A., Sharma, G., Pillai, B., and Sengupta, S. (2006) Biochem. J. 396, 61-69), the str4Δ strain did not exhibit an endoplasmic reticulum stress response. This suggests that homocysteine induces varied response depending on the flux of homocysteine metabolism. We also observed altered expression of mitochondrial genes, defective membrane potential, and fragmentation of the mitochondrial network together with the increased expression of fission genes indicating that the imbalance in homocysteine metabolism has a major effect on mitochondrial functions. Furthermore, treatment of cells with homocysteine or AdoHcy resulted in apoptosis as revealed by annexin V staining and TUNEL assay. Cumulatively, our results suggest that elevated levels of homocysteine lead to mitochondrial dysfunction, which could potentially initiate pro-apoptotic pathways, and this could be one of the mechanisms underlying homocysteine-induced pathogenicity.

Toll-like receptor 2 and DC-SIGNR1 differentially regulate suppressors of cytokine signaling 1 in dendritic cells during Mycobacterium tuberculosis infection.

A hallmark of protective immunity during Mycobacterium tuberculosis (M. tb) infection is the regulated secretion of pro-inflammatory and regulatory cytokines. Suppressors of Cytokine Signaling (SOCS) are key regulators of cytokine secretion and function. In this study we investigated regulation of Toll-like receptor 2 (TLR2) and dendritic cell-specific ICAM-3 grabbing non-integrin receptor 1 (DC-SIGNR1)-mediated SOCS1 expression in DCs during M. tb infection. We show that, compared with TLR2, stimulating DC-SIGNR1 on DCs induces higher SOCS1 expression and lower interleukin-12 production. Co-stimulating DC-SIGNR1 and TLR2 differentially regulates SOCS1 expression depending on the relative concentration of their ligands. Stimulating DC-SIGNR1 with M. tb infection increases SOCS1 expression, while stimulating TLR2 with M. tb infection reduces SOCS1 expression. Knockdown of SOCS1 in DCs by siRNA enhances interleukin-12 transcription and protein expression upon DC-SIGNR1 stimulation. Raf-1 and Syk differentially regulate TLR2- and DC-SIGNR1-mediated SOCS1 expression. In addition, DC-SIGNR1 shows greater association with SOCS1 when compared with TLR2. Interestingly, compared with healthy asymptomatic individuals, peripheral blood mononuclear cells of patients with active tuberculosis disease showed higher expression of SOCS1, which was reduced following chemotherapy. Similarly, stimulating DC-SIGNR1 on DCs from M. tb-infected TLR2(-/-) mice enhanced SOCS1 expression that was reduced following chemotherapy. Further, knockdown of SOCS1 in mouse DCs or human peripheral blood mononuclear cells resulted in increased killing of virulent M. tb. These results indicate that TLR2 and DC-SIGNR1 differentially regulate SOCS1 expression during M. tb infection. This in turn regulates M. tb survival by governing key cytokine expression.

Compound loss of muscleblind-like function in myotonic dystrophy.

Myotonic dystrophy (DM) is a multi-systemic disease that impacts cardiac and skeletal muscle as well as the central nervous system (CNS). DM is unusual because it is an RNA-mediated disorder due to the expression of toxic microsatellite expansion RNAs that alter the activities of RNA processing factors, including the muscleblind-like (MBNL) proteins. While these mutant RNAs inhibit MBNL1 splicing activity in heart and skeletal muscles, Mbnl1 knockout mice fail to recapitulate the full-range of DM symptoms in these tissues. Here, we generate mouse Mbnl compound knockouts to test the hypothesis that Mbnl2 functionally compensates for Mbnl1 loss. Although Mbnl1(-/-) ; Mbnl2(-/-) double knockouts (DKOs) are embryonic lethal, Mbnl1(-/-) ; Mbnl2(+/-) mice are viable but develop cardinal features of DM muscle disease including reduced lifespan, heart conduction block, severe myotonia and progressive skeletal muscle weakness. Mbnl2 protein levels are elevated in Mbnl1(-/-) knockouts where Mbnl2 targets Mbnl1-regulated exons. These findings support the hypothesis that compound loss of MBNL function is a critical event in DM pathogenesis and provide novel mouse models to investigate additional pathways disrupted in this RNA-mediated disease.