Human Pumilio proteins, PUM1 and PUM2, are sequence specific RNA-binding proteins that regulate protein expression. We used RNA-seq, rigorous statistical testing and an experimentally derived fold change cut-off to identify nearly 1000 target RNAs—including mRNAs and non-coding RNAs—that are functionally regulated by PUMs. Bioinformatic analysis defined a PUM Response Element (PRE) that was significantly enriched in transcripts that increased in abundance and matches the PUM RNA-binding consensus. We created a computational model that incorporates PRE position and frequency within an RNA relative to the magnitude of regulation. The model reveals significant correlation of PUM regulation with PREs in 3′ untranslated regions (UTRs), coding sequences and non-coding RNAs, but not 5′ UTRs. To define direct, high confidence PUM targets, we cross-referenced PUM-regulated RNAs with all PRE-containing RNAs and experimentally defined PUM-bound RNAs. The results define nearly 300 direct targets that include both PUM-repressed and, surprisingly, PUM-activated target RNAs. Annotation enrichment analysis reveal that PUMs regulate genes from multiple signaling pathways and developmental and neurological processes. Moreover, PUM target mRNAs impinge on human disease genes linked to cancer, neurological disorders and cardiovascular disease. These discoveries pave the way for determining how the PUM-dependent regulatory network impacts biological functions and disease states.

Purpose Androgen receptor (AR) variant AR-V7 is a ligand-independent transcription factor that promotes prostate cancer resistance to AR-targeted therapies. Accordingly, efforts are underway to develop strategies for monitoring and inhibiting AR-V7 in castration-resistant prostate cancer (CRPC). The purpose of this study was to understand whether other AR variants may be co-expressed with AR-V7 and promote resistance to AR-targeted therapies. Experimental Design We utilized complementary short- and long-read sequencing of intact AR mRNA isoforms to characterize AR expression in CRPC models. Co-expression of AR-V7 and AR-V9 mRNA in CRPC metastases and circulating tumor cells was assessed by RNA-seq and RT-PCR, respectively. Expression of AR-V9 protein in CRPC models was evaluated with polyclonal antisera. Multivariate analysis was performed to test whether AR variant mRNA expression in metastatic tissues was associated with a 12-week progression-free survival endpoint in a prospective clinical trial of 78 CRPC-stage patients initiating therapy with the androgen synthesis inhibitor, abiraterone acetate. Results AR-V9 was frequently co-expressed with AR-V7. Both AR variant species were found to share a common 3’ terminal cryptic exon, which rendered AR-V9 susceptible to experimental manipulations that were previously-thought to target AR-V7 uniquely. AR-V9 promoted ligand-independent growth of prostate cancer cells. High AR-V9 mRNA expression in CRPC metastases was predictive of primary resistance to abiraterone acetate (HR = 4.0, 95% CI = 1.31–12.2, P = 0.02). Conclusions AR-V9 may be an important component of therapeutic resistance in CRPC.

Palmitoylation is the post-translational addition of a palmitate moiety to a cysteine residue through a covalent thioester bond. The addition and removal of this modification is controlled by both palmitoyl acyl-transferases and thioesterases. Using bioinformatic analysis, we identified 22 DHHC family palmitoyl acyl-transferase homologs in the Drosophila genome. We used in situ hybridization, RT-PCR, and published FlyAtlas microarray data to characterize the expression patterns of all 22 fly homologs. Our results indicate that all are expressed genes, but several, including CG1407, CG4676, CG5620, CG6017/dHIP14, CG6618, CG6627, and CG17257 appear to be enriched in neural tissues suggesting that they are important for neural function. Furthermore, we have found that several may be expressed in a sex-specific manner with adult male-specific expression of CG4483 and CG17195. Using tagged versions of the DHHC genes, we demonstrate that fly DHHC proteins are primarily located in either the Golgi Apparatus or Endoplasmic Reticulum in S2 cells, except for CG1407, which was found on the plasma membrane. We also characterized the subcellular localization and expression of the three known thioesterases: Palmitoyl-protein Thioesterase 1 (Ppt1), Palmitoyl-protein Thioesterase 2 (Ppt2), and Acyl-protein Thioesterase 1 (APT1). Our results indicate that Ppt1 and Ppt2 are the major lysosomal thioesterases while APT1 is the likely cytoplasmic thioesterase. Finally, in vivo rescue experiments show that Ppt2 expression cannot rescue the neural inclusion phenotypes associated with loss of Ppt1, further supporting distinct functions and substrates for these two thioesterases. These results will serve as the basis for a more complete understanding of the protein palmitoylome's normal cellular functions in the fly and will lead to further insights into the molecular etiology of diseases associated with the mis-regulation of palmitoylation.

Prostate cancer is the second leading cause of male cancer deaths due to disease progression to castration resistant prostate cancer (CRPC). Androgen receptor (AR) splice variants including AR-V7 function as constitutively active transcription factors in CRPC cells, thereby promoting resistance to AR-targeted therapies. To date, there are no AR variant specific treatments for CRPC. Here we report that the splicing of AR variants AR-V7 as well as AR-V1 and AR-V9 is regulated coordinately by a single polyadenylation signal in AR intron 3. Blocking this signal with morpholino technology or silencing of the polyadenylation factor CPSF1 caused a splice switch that inhibited expression of AR variants and blocked androgen-independent growth of CRPC cells. Our findings support the development of new therapies targeting the polyadenylation signal in AR intron 3 as a strategy to prevent expression of a broad array of AR variants in CRPC.

Metastatic disease is responsible for the majority of prostate cancer deaths. The standard treatment for metastatic disease is surgical or chemical castration in the form of androgen deprivation therapy. Despite initial success and disease regression, resistance to therapy ultimately develops and the disease transitions to castration resistant prostate cancer, which is uniformly fatal. Thus, developing an understanding of genetic evolution in metastasis and in response to therapy has been a focus of recent studies. Large-scale sequencing studies have provided an expansive catalog of the mutation events that occur in the prostate cancer genome at various stages of disease progression. Smaller-scale studies have interrogated the genomic composition of multiple metastatic sites within individual patients, or have tracked clonal evolution longitudinally in tissues, circulating tumor cells, or circulating tumor DNA. Collectively, these efforts have provided a new conceptual framework for understanding the origin of prostate cancer, as well as the origin and evolution of metastatic disease. In this review, we will highlight these recent insights into the spatiotemporal landscape of genetic evolution of prostate cancer.

BackgroundInsertions and deletions (indels) are a major class of genomic variation associated with human disease. Indels are primarily detected from DNA sequencing (DNA-seq) data but their transcriptional consequences remain unexplored due to challenges in discriminating medium-sized and large indels from splicing events in RNA-seq data.ResultsHere, we developed transIndel, a splice-aware algorithm that parses the chimeric alignments predicted by a short read aligner and reconstructs the mid-sized insertions and large deletions based on the linear alignments of split reads from DNA-seq or RNA-seq data. TransIndel exhibits competitive or superior performance over eight state-of-the-art indel detection tools on benchmarks using both synthetic and real DNA-seq data. Additionally, we applied transIndel to DNA-seq and RNA-seq datasets from 333 primary prostate cancer patients from The Cancer Genome Atlas (TCGA) and 59 metastatic prostate cancer patients from AACR-PCF Stand-Up- To-Cancer (SU2C) studies. TransIndel enhanced the taxonomy of DNA- and RNA-level alterations in prostate cancer by identifying recurrent FOXA1 indels as well as exitron splicing in genes implicated in disease progression.ConclusionsOur study demonstrates that transIndel is a robust tool for elucidation of medium- and large-sized indels from DNA-seq and RNA-seq data. Including RNA-seq in indel discovery efforts leads to significant improvements in sensitivity for identification of med-sized and large indels missed by DNA-seq, and reveals non-canonical RNA-splicing events in genes associated with disease pathology.Electronic supplementary materialThe online version of this article (10.1186/s12864-018-4671-4) contains supplementary material, which is available to authorized users.

<p>This file contains supplementary materials and methods including qPCR primer sequences, 2C subcellular fractionation, and additional details for RNA-Seq analyses.</p>

<p>This file contains supplementary figures S1-S10 and corresponding figure legends. Figure S1: TALEN activity and genetic correction of 22Rv1 sub-lines. Figure S2: 5' and 3' breakpoint sequence junctions of 22Rv1 undup1-3 sub-lines. Figure S3: AR subcellular localization in 22Rv1-undup3 cell line. Figure S4: AR target gene expression panel in 22Rv1 and gene corrected sublines. Figure S5: Knockdown of splicing factors U2AF2, SRSF1, and PTBP1. Figure S6: Blockade of the AR CE3 PAS blocks AR-V expression in LNCaP and VCaP cells. Figure S7: Knockdown of CE3 in 22Rv1 and VCaP cells. Figure S8: Mutual exclusivity matrix depicting co-occurrence of alterations in the core CPSF complex. Figure S9: Confirmation of CPSF complex gene knockdown. Figure S10: Knockdown of CPSF1 in LNCaP95 cells.</p>