Lineage-specific splicing regulation of MAPT gene in the primate brain.

Divergence of precursor messenger RNA (pre-mRNA) alternative splicing (AS) is widespread in mammals, including primates, but the underlying mechanisms and functional impact are poorly understood. Here, we modeled cassette exon inclusion in primate brains as a quantitative trait and identified 1,170 (∼3%) exons with lineage-specific splicing shifts under stabilizing selection. Among them, microtubule-associated protein tau (MAPT) exons 2 and 10 underwent anticorrelated, two-step evolutionary shifts in the catarrhine and hominoid lineages, leading to their present inclusion levels in humans. The developmental-stage-specific divergence of exon 10 splicing, whose dysregulation can cause frontotemporal lobar degeneration (FTLD), is mediated by divergent distal intronic MBNL-binding sites. Competitive binding of these sites by CRISPR-dCas13d/gRNAs effectively reduces exon 10 inclusion, potentially providing a therapeutically compatible approach to modulate tau isoform expression. Our data suggest adaptation of MAPT function and, more generally, a role for AS in the evolutionary expansion of the primate brain.

Depletion of HuR in murine skeletal muscle enhances exercise endurance and prevents cancer-induced muscle atrophy.

The master posttranscriptional regulator HuR promotes muscle fiber formation in cultured muscle cells. However, its impact on muscle physiology and function in vivo is still unclear. Here, we show that muscle-specific HuR knockout (muHuR-KO) mice have high exercise endurance that is associated with enhanced oxygen consumption and carbon dioxide production. muHuR-KO mice exhibit a significant increase in the proportion of oxidative type I fibers in several skeletal muscles. HuR mediates these effects by collaborating with the mRNA decay factor KSRP to destabilize the PGC-1α mRNA. The type I fiber-enriched phenotype of muHuR-KO mice protects against cancer cachexia-induced muscle loss. Therefore, our study uncovers that under normal conditions HuR modulates muscle fiber type specification by promoting the formation of glycolytic type II fibers. We also provide a proof-of-principle that HuR expression can be targeted therapeutically in skeletal muscles to combat cancer-induced muscle wasting.

Proteomic analysis reveals that wildtype and alanine-expanded nuclear poly(A)-binding protein exhibit differential interactions in skeletal muscle.

Oculopharyngeal muscular dystrophy (OPMD) is a late-onset, primarily autosomal dominant disease caused by a short GCN expansion in the PABPN1 (polyadenylate-binding protein nuclear 1) gene that results in an alanine expansion at the N terminus of the PABPN1 protein. Expression of alanine-expanded PABPN1 is linked to the formation of nuclear aggregates in tissues from individuals with OPMD. However, as with other nuclear aggregate-associated diseases, controversy exists over whether these aggregates are the direct cause of pathology. An emerging hypothesis is that a loss of PABPN1 function and/or aberrant protein interactions contribute to pathology in OPMD. Here, we present the first global proteomic analysis of the protein interactions of WT and alanine-expanded PABPN1 in skeletal muscle tissue. These data provide both insight into the function of PABPN1 in muscle and evidence that the alanine expansion alters the protein-protein interactions of PABPN1. We extended this analysis to demonstrate altered complex formation with and loss of function of TDP-43 (TAR DNA-binding protein 43), which we show interacts with alanine-expanded but not WT PABPN1. The results from our study support a model where altered protein interactions with alanine-expanded PABPN1 that lead to loss or gain of function could contribute to pathology in OPMD.

Post-transcriptional regulation of Pabpn1 by the RNA binding protein HuR.

RNA processing is critical for proper spatial and temporal control of gene expression. The ubiquitous nuclear polyadenosine RNA binding protein, PABPN1, post-transcriptionally regulates multiple steps of gene expression. Mutations in the PABPN1 gene expanding an N-terminal alanine tract in the PABPN1 protein from 10 alanines to 11-18 alanines cause the muscle-specific disease oculopharyngeal muscular dystrophy (OPMD), which affects eyelid, pharynx, and proximal limb muscles. Previous work revealed that the Pabpn1 transcript is unstable, contributing to low steady-state Pabpn1 mRNA and protein levels in vivo, specifically in skeletal muscle, with even lower levels in muscles affected in OPMD. Thus, low levels of PABPN1 protein could predispose specific tissues to pathology in OPMD. However, no studies have defined the mechanisms that regulate Pabpn1 expression. Here, we define multiple cis-regulatory elements and a trans-acting factor, HuR, which regulate Pabpn1 expression specifically in mature muscle in vitro and in vivo. We exploit multiple models including C2C12 myotubes, primary muscle cells, and mice to determine that HuR decreases Pabpn1 expression. Overall, we have uncovered a mechanism in mature muscle that negatively regulates Pabpn1 expression in vitro and in vivo, which could provide insight to future studies investigating therapeutic strategies for OPMD treatment.

Novel mouse models of oculopharyngeal muscular dystrophy (OPMD) reveal early onset mitochondrial defects and suggest loss of PABPN1 may contribute to pathology.

Oculopharyngeal muscular dystrophy (OPMD) is a late onset disease caused by polyalanine expansion in the poly(A) binding protein nuclear 1 (PABPN1). Several mouse models have been generated to study OPMD; however, most of these models have employed transgenic overexpression of alanine-expanded PABPN1. These models do not recapitulate the OPMD patient genotype and PABPN1 overexpression could confound molecular phenotypes. We have developed a knock-in mouse model of OPMD (Pabpn1+/A17) that contains one alanine-expanded Pabpn1 allele under the control of the native promoter and one wild-type Pabpn1 allele. This mouse is the closest available genocopy of OPMD patients. We show that Pabpn1+/A17 mice have a mild myopathic phenotype in adult and aged animals. We examined early molecular and biochemical phenotypes associated with expressing native levels of A17-PABPN1 and detected shorter poly(A) tails, modest changes in poly(A) signal (PAS) usage, and evidence of mitochondrial damage in these mice. Recent studies have suggested that a loss of PABPN1 function could contribute to muscle pathology in OPMD. To investigate a loss of function model of pathology, we generated a heterozygous Pabpn1 knock-out mouse model (Pabpn1+/Δ). Like the Pabpn1+/A17 mice, Pabpn1+/Δ mice have mild histologic defects, shorter poly(A) tails, and evidence of mitochondrial damage. However, the phenotypes detected in Pabpn1+/Δ mice only partially overlap with those detected in Pabpn1+/A17 mice. These results suggest that loss of PABPN1 function could contribute to but may not completely explain the pathology detected in Pabpn1+/A17 mice.

Pharyngeal Satellite Cells Undergo Myogenesis Under Basal Conditions and Are Required for Pharyngeal Muscle Maintenance.

The pharyngeal muscles of the nasal, oral, and laryngeal pharynxes are required for swallowing. Pharyngeal muscles are preferentially affected in some muscular dystrophies yet spared in others. Muscle stem cells, called satellite cells, may be critical factors in the development of pharyngeal muscle disorders; however, very little is known about pharyngeal satellite cells (PSC) and their role in pharyngeal muscles. We show that PSC are distinct from the commonly studied hindlimb satellite cells both transcriptionally and biologically. Under basal conditions PSC proliferate, progress through myogenesis, and fuse with pharyngeal myofibers. Furthermore, PSC exhibit biologic differences dependent on anatomic location in the pharynx. Importantly, PSC are required to maintain myofiber size and myonuclear number in pharyngeal myofibers. Together, these results demonstrate that PSC are critical for pharyngeal muscle maintenance and suggest that satellite cell impairment could contribute to pharyngeal muscle pathology associated with various muscular dystrophies and aging.

miR-375 activates p21 and suppresses telomerase activity by coordinately regulating HPV E6/E7, E6AP, CIP2A, and 14-3-3ζ.

BACKGROUND: While microRNAs (miRNAs) are extensively studied in post-transcriptional regulation of gene expressions in many biological processes, cellular miRNA-mediated regulation of viral genes remains unclear. In particular, the interplay between human papillomavirus (HPV) genes and miRNAs and how these interactions contribute to HPV-associated cancers remain elusive. METHODS: Transient transfection of miR-375-mimic was used to compensate the loss-of-function of miR-375 in HPV-positive cancer. Regulation of oncogenic molecules and their downstream molecules via miR-375 in HPV-positive cancer was investigated using qRT-PCR, western blot, dual luciferase assay, indirect immunofluorescence analysis. All experiments were conducted at least three times to achieve statistical significance determined by Student t-test. RESULTS: In this study, we demonstrated how miR-375 negatively regulates HPV16 and 18 transcripts. We also found a cellular protein, E6-associated protein (E6AP), directly regulated by miR-375. miR-375-mediated repression of HPV transcripts and E6AP elevated major tumor suppressors p53, p21, and retinoblastoma protein 1 (RB). Cooperative regulation of miR-375 targets along with the increase of tumor suppressors led to ~60% reduction of telomerase reverse transcriptase (TERT) transcription followed by ~35% decrease of telomerase activity. Furthermore, miR-375-mediated regulation of 14-3-3ζ contributes to decrease telomerase activity by altering nuclear translocation of TERT. CONCLUSION: Taken together, miR-375-mediated suppression of multiple oncogenic components in HPV-associated carcinogenesis generates a cumulative biological response to rescue key tumor suppressors and diminish telomerase activity, which results in cell cycle arrest and cell proliferation inhibition.

Tumor suppressor miR-375 regulates MYC expression via repression of CIP2A coding sequence through multiple miRNA-mRNA interactions.

MicroRNAs (miRNAs) are small, noncoding RNAs involved in posttranscriptional regulation of protein-coding genes in various biological processes. In our preliminary miRNA microarray analysis, miR-375 was identified as the most underexpressed in human oral tumor versus controls. The purpose of the present study is to examine the function of miR-375 as a candidate tumor suppressor miRNA in oral cancer. Cancerous inhibitor of PP2A (CIP2A), a guardian of oncoprotein MYC, is identified as a candidate miR-375 target based on bioinformatics. Luciferase assay accompanied by target sequence mutagenesis elucidates five functional miR-375-binding sites clustered in the CIP2A coding sequence close to the C-terminal domain. Overexpression of CIP2A is clearly demonstrated in oral cancers, and inverse correlation between miR-375 and CIP2A is observed in the tumors, as well as in NCI-60 cell lines, indicating the potential generalized involvement of the miR-375-CIP2A relationship in many other cancers. Transient transfection of miR-375 in oral cancer cells reduces the expression of CIP2A, resulting in decrease of MYC protein levels and leading to reduced proliferation, colony formation, migration, and invasion. Therefore this study shows that underexpression of tumor suppressor miR-375 could lead to uncontrolled CIP2A expression and extended stability of MYC, which contributes to promoting cancerous phenotypes.

Keratinization-associated miR-7 and miR-21 regulate tumor suppressor reversion-inducing cysteine-rich protein with kazal motifs (RECK) in oral cancer.

MicroRNAs (miRNAs) are small non-coding RNAs that posttranscriptionally regulate gene expression during many biological processes. Recently, the aberrant expressions of miRNAs have become a major focus in cancer research. The purpose of this study was to identify deregulated miRNAs in oral cancer and further focus on specific miRNAs that were related to patient survival. Here, we report that miRNA expression profiling provided more precise information when oral squamous cell carcinomas were subcategorized on the basis of clinicopathological parameters (tumor primary site, histological subtype, tumor stage, and HPV16 status). An innovative radar chart analysis method was developed to depict subcategories of cancers taking into consideration the expression patterns of multiple miRNAs combined with the clinicopathological parameters. Keratinization of tumors and the high expression of miR-21 were the major factors related to the poor prognosis of patients. Interestingly, a majority of the keratinized tumors expressed high levels of miR-21. Further investigations demonstrated the regulation of the tumor suppressor gene reversion-inducing cysteine-rich protein with kazal motifs (RECK) by two keratinization-associated miRNAs, miR-7 and miR-21. Transfection of miR-7 and miR-21-mimics reduced the expression of RECK through direct miRNA-mediated regulation, and these miRNAs were inversely correlated with RECK in CAL 27 orthotopic xenograft tumors. Furthermore, a similar inverse correlation was demonstrated in CAL 27 cells treated in vitro by different external stimuli such as trypsinization, cell density, and serum concentration. Taken together, our data show that keratinization is associated with poor prognosis of oral cancer patients and keratinization-associated miRNAs mediate deregulation of RECK which may contribute to the aggressiveness of tumors.