Preferential binding of a stable G3BP ribonucleoprotein complex to intron-retaining transcripts in mouse brain and modulation of their expression in the cerebellum.

Neuronal granules play an important role in the localization and transport of translationally silenced messenger ribonucleoproteins in neurons. Among the factors associated with these granules, the RNA-binding protein G3BP1 (stress-granules assembly factor) is involved in neuronal plasticity and is induced in Alzheimer's disease. We immunopurified a stable complex containing G3BP1 from mouse brain and performed high-throughput sequencing and cross-linking immunoprecipitation to identify the associated RNAs. The G3BP-complex contained the deubiquitinating protease USP10, CtBP1 and the RNA-binding proteins Caprin-1, G3BP2a and splicing factor proline and glutamine rich, or PSF. The G3BP-complex binds preferentially to transcripts that retain introns, and to non-coding sequences like 3'-untranslated region and long non-coding RNAs. Specific transcripts with retained introns appear to be enriched in the cerebellum compared to the rest of the brain and G3BP1 depletion decreased this intron retention in the cerebellum of G3BP1 knockout mice. Among the enriched transcripts, we found an overrepresentation of genes involved in synaptic transmission, especially glutamate-related neuronal transmission. Notably, G3BP1 seems to repress the expression of the mature Grm5 (metabotropic glutamate receptor 5) transcript, by promoting the retention of an intron in the immature transcript in the cerebellum. Our results suggest that G3BP is involved in a new functional mechanism to regulate non-coding RNAs including intron-retaining transcripts, and thus have broad implications for neuronal gene regulation, where intron retention is widespread.

Canadian anaplastic lymphoma kinase study: a model for multicenter standardization and optimization of ALK testing in lung cancer.

INTRODUCTION: Fluorescence in situ hybridization (FISH) is currently the standard for diagnosing anaplastic lymphoma kinase (ALK)-rearranged (ALK+) lung cancers for ALK inhibitor therapies. ALK immunohistochemistry (IHC) may serve as a screening and alternative diagnostic method. The Canadian ALK (CALK) study was initiated to implement a multicenter optimization and standardization of laboratory developed ALK IHC and FISH tests across 14 hospitals. METHODS: Twenty-eight lung adenocarcinomas with known ALK status were used as blinded study samples. Thirteen laboratories performed IHC using locally developed staining protocols for 5A4, ALK1, or D5F3 antibodies; results were assessed by H-score. Twelve centers conducted FISH using protocols based on Vysis' ALK break-apart FISH kit. Initial IHC results were used to optimize local IHC protocols, followed by a repeat IHC study to assess the results of standardization. Three laboratories conducted a prospective parallel IHC and FISH analysis on 411 consecutive clinical samples using post-validation optimized assays. RESULTS: Among study samples, FISH demonstrated 22 consensus ALK+ and six ALK wild type tumors. Preoptimization IHC scores from 12 centers with 5A4 and the percent abnormal cells by FISH from 12 centers showed intraclass correlation coefficients of 0.83 and 0.68, respectively. IHC optimization improved the intraclass correlation coefficients to 0.94. Factors affecting FISH scoring and outliers were identified. Post-optimization concurrent IHC/FISH testing in 373 informative cases revealed 100% sensitivity and specificity for IHC versus FISH. CONCLUSIONS: Multicenter standardization study may accelerate the implementation of ALK testing protocols across a country/region. Our data support the use of an appropriately validated IHC assay to screen for ALK+ lung cancers.