Human Staufen1 (Stau1) is a double-stranded RNA (dsRNA)-binding protein implicated in multiple post-transcriptional gene-regulatory processes. Here we combined RNA immunoprecipitation in tandem (RIPiT) with RNase footprinting, formaldehyde cross-linking, sonication-mediated RNA fragmentation and deep sequencing to map Staufen1-binding sites transcriptome wide. We find that Stau1 binds complex secondary structures containing multiple short helices, many of which are formed by inverted Alu elements in annotated 3′ untranslated regions (UTRs) or in 'strongly distal' 3′ UTRs. Stau1 also interacts with actively translating ribosomes and with mRNA coding sequences (CDSs) and 3′ UTRs in proportion to their GC content and propensity to form internal secondary structure. On mRNAs with high CDS GC content, higher Stau1 levels lead to greater ribosome densities, thus suggesting a general role for Stau1 in modulating translation elongation through structured CDS regions. Our results also indicate that Stau1 regulates translation of transcription-regulatory proteins.Staufen proteins are highly conserved dsRNA-binding proteins (dsRBPs) found in most bilateral animals 1 . Mammals contain two Staufen paralogs encoded by different loci. Stau1, Reprints and permissions information is available online at
Fully assembled ribosomes exist in two populations: polysomes and monosomes. While the former has been studied extensively, to what extent translation occurs on monosomes and its importance for overall translational output remain controversial. Here, we used ribosome profiling to examine the translational status of 80S monosomes in Saccharomyces cerevisiae. We found that the vast majority of 80S monosomes are elongating, not initiating. Further, most mRNAs exhibit some degree of monosome occupancy, with monosomes predominating on nonsense-mediated decay (NMD) targets, upstream open reading frames (uORFs), canonical ORFs shorter than ∼ 590 nt, and ORFs for which the total time required to complete elongation is substantially shorter than that required for initiation. Importantly, mRNAs encoding low-abundance regulatory proteins tend to be enriched in the monosome fraction. Our data highlight the importance of monosomes for the translation of highly regulated mRNAs.
Fusion genes are a major cause of cancer. Their rapid and accurate diagnosis can inform clinical action, but current molecular diagnostic assays are restricted in resolution and throughput. Here, we show that targeted RNA sequencing (RNAseq) can overcome these limitations. First, we establish that fusion gene detection with targeted RNAseq is both sensitive and quantitative by optimising laboratory and bioinformatic variables using spike-in standards and cell lines. Next, we analyse a clinical patient cohort and improve the overall fusion gene diagnostic rate from 63% with conventional approaches to 76% with targeted RNAseq while demonstrating high concordance for patient samples with previous diagnoses. Finally, we show that targeted RNAseq offers additional advantages by simultaneously measuring gene expression levels and profiling the immune-receptor repertoire. We anticipate that targeted RNAseq will improve clinical fusion gene detection, and its increasing use will provide a deeper understanding of fusion gene biology.
Deep sequencing of strand-specific cDNA libraries is now a ubiquitous tool for identifying and quantifying RNAs in diverse sample types. The accuracy of conclusions drawn from these analyses depends on precise and quantitative conversion of the RNA sample into a DNA library suitable for sequencing. Here, we describe an optimized method of preparing strand-specific RNA deep sequencing libraries from small RNAs and variably sized RNA fragments obtained from ribonucleoprotein particle footprinting experiments or fragmentation of long RNAs. Our approach works across a wide range of input amounts (400 pg to 200 ng), is easy to follow and produces a library in 2–3 days at relatively low reagent cost, all while giving the user complete control over every step. Because all enzymatic reactions were optimized and driven to apparent completion, sequence diversity and species abundance in the input sample are well preserved.
PURPOSE Before anaplastic lymphoma kinase (ALK) inhibitors, treatment options for ALK-positive inflammatory myofibroblastic tumors (AP-IMTs) were unsatisfactory. We retrospectively analyzed the outcome of patients with AP-IMT treated with crizotinib to document response, toxicity, survival, and features associated with relapse. METHODS The cohort comprised eight patients with AP-IMT treated with crizotinib and surgery. Outcome measures were progression-free and overall survival after commencing crizotinib, treatment-related toxicities, features associated with relapse, outcome after relapse, and outcome after ceasing crizotinib. RESULTS The median follow-up after commencing crizotinib was 3 years (range, 0.9 to 5.5 years). The major toxicity was neutropenia. All patients responded to crizotinib. Five were able to discontinue therapy without recurrence (median treatment duration, 1 year; range, 0.2 to 3.0 years); one continues on crizotinib. Two critically ill patients with initial complete response experienced relapse while on therapy. Both harbored RANBP2-ALK fusions and responded to alternative ALK inhibitors; one ultimately died as a result of progressive disease, whereas the other remains alive on treatment. Progression-free and overall survival since commencement of crizotinib is 0.75 ± 0.15% and 0.83 ± 0.15%, respectively. CONCLUSION We confirm acceptable toxicity and excellent disease control in patients with AP-IMT treated with crizotinib, which may be ceased without recurrence in most. Relapses occurred in two of three patients with RANBP2-ALK translocated IMT, which suggests that such patients require additional therapy.
Despite intensive multimodal treatment of sarcomas, a heterogeneous group of malignant tumors arising from connective tissue, survival remains poor. Candidate-based targeted treatments have demonstrated limited clinical success, urging an unbiased and comprehensive analysis of oncogenic signaling networks to reveal therapeutic targets and personalized treatment strategies. Here we applied mass spectrometry-based phosphoproteomic profiling to the largest and most heterogeneous set of sarcoma cell lines characterized to date and identified novel tyrosine phosphorylation patterns, enhanced tyrosine kinases in specific subtypes, and potential driver kinases. ALK was identified as a novel driver in the Aska-SS synovial sarcoma (SS) cell line via expression of an ALK variant with a large extracellular domain deletion (ALK D2-17 ). Functional ALK dependency was confirmed in vitro and in vivo with selective inhibitors. Importantly, ALK immunopositivity was detected in 6 of 43 (14%) of SS patient specimens, one of which exhibited an ALK rearrangement. High PDGFRa phosphorylation also characterized SS cell lines, which was accompanied by enhanced MET activation in Yamato-SS cells. Although Yamato-SS cells were sensitive to crizotinib (ALK/MET-inhibitor) but not pazopanib (VEGFR/PDGFR-inhibitor) monotherapy in vitro, synergistic effects were observed upon drug combination. In vivo, both drugs were individually effective, with pazopanib efficacy likely attributable to reduced angiogenesis. MET or PDGFRa expression was detected in 58% and 84% of SS patients, respectively, with coexpression in 56%. Consequently, our integrated approach has led to the identification of ALK and MET as promising therapeutic targets in SS.
Background The androgen‐regulated gene TMPRSS2 to the ETS transcription factor gene ERG fusion is the most common genomic alteration acquired during prostate tumorigenesis and biased toward men of European ancestry. In contrast, African American men present with more advanced disease, yet their tumors are less likely to acquire TMPRSS2‐ERG . Data for Africa is scarce. Methods RNA was made available for genomic analyses from 181 prostate tissue biopsy cores from Black South African men, 94 with and 87 without pathological evidence for prostate cancer. Reverse transcription polymerase chain reaction was used to screen for the TMPRSS2‐ERG fusion, while transcript junction coordinates and isoform frequencies, including novel gene fusions, were determined using targeted RNA sequencing. Results Here we report a frequency of 13% for TMPRSS2‐ERG in tumors from Black South Africans. Present in 12/94 positive versus 1/87 cancer negative prostate tissue cores, this suggests a 92.62% predictivity for a positive cancer diagnosis ( P = 0.0031). At a frequency of almost half that reported for African Americans and roughly a quarter of that reported for men of European ancestry, acquisition of TMPRSS2‐ERG appears to be inversely associated with aggressive prostate cancer. Further support was provided by linking the presence of TMPRSS2‐ERG to low‐grade disease in younger patients ( P = 0.0466), with higher expressing distal ERG fusion junction coordinates. Conclusions Only the second study of its kind for the African continent, we support a link between TMPRSS2‐ERG status and prostate cancer racial health disparity beyond the borders of the United States. We call for urgent evaluation of androgen deprivation therapy within Africa.
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