PRPF mutations are associated with generalized defects in spliceosome formation and pre-mRNA splicing in patients with retinitis pigmentosa.

Proteins PRPF31, PRPF3 and PRPF8 (RP-PRPFs) are ubiquitously expressed components of the spliceosome, a macromolecular complex that processes nearly all pre-mRNAs. Although these spliceosomal proteins are conserved in eukaryotes and are essential for survival, heterozygous mutations in human RP-PRPF genes lead to retinitis pigmentosa, a hereditary disease restricted to the eye. Using cells from patients with 10 different mutations, we show that all clinically relevant RP-PRPF defects affect the stoichiometry of spliceosomal small nuclear RNAs (snRNAs), the protein composition of tri-small nuclear ribonucleoproteins and the kinetics of spliceosome assembly. These mutations cause inefficient splicing in vitro and affect constitutive splicing ex-vivo by impairing the removal of at least 9% of endogenously expressed introns. Alternative splicing choices are also affected when RP-PRPF defects are present. Furthermore, we show that the steady-state levels of snRNAs and processed pre-mRNAs are highest in the retina, indicating a particularly elevated splicing activity. Our results suggest a role for PRPFs defects in the etiology of PRPF-linked retinitis pigmentosa, which appears to be a truly systemic splicing disease. Although these mutations cause widespread and important splicing defects, they are likely tolerated by the majority of human tissues but are critical for retinal cell survival.

Circadian and feeding rhythms differentially affect rhythmic mRNA transcription and translation in mouse liver.

Diurnal oscillations of gene expression are a hallmark of rhythmic physiology across most living organisms. Such oscillations are controlled by the interplay between the circadian clock and feeding rhythms. Although rhythmic mRNA accumulation has been extensively studied, comparatively less is known about their transcription and translation. Here, we quantified simultaneously temporal transcription, accumulation, and translation of mouse liver mRNAs under physiological light-dark conditions and ad libitum or night-restricted feeding in WT and brain and muscle Arnt-like 1 (Bmal1)-deficient animals. We found that rhythmic transcription predominantly drives rhythmic mRNA accumulation and translation for a majority of genes. Comparison of wild-type and Bmal1 KO mice shows that circadian clock and feeding rhythms have broad impact on rhythmic gene expression, Bmal1 deletion affecting surprisingly both transcriptional and posttranscriptional levels. Translation efficiency is differentially regulated during the diurnal cycle for genes with 5'-Terminal Oligo Pyrimidine tract (5'-TOP) sequences and for genes involved in mitochondrial activity, many harboring a Translation Initiator of Short 5'-UTR (TISU) motif. The increased translation efficiency of 5'-TOP and TISU genes is mainly driven by feeding rhythms but Bmal1 deletion also affects amplitude and phase of translation, including TISU genes. Together this study emphasizes the complex interconnections between circadian and feeding rhythms at several steps ultimately determining rhythmic gene expression and translation.

DNA Topoisomerase I Gene Copy Number and mRNA Expression Assessed as Predictive Biomarkers for Adjuvant Irinotecan in Stage II/III Colon Cancer.

PURPOSE: Prospective-retrospective assessment of theTOP1gene copy number andTOP1mRNA expression as predictive biomarkers for adjuvant irinotecan in stage II/III colon cancer. EXPERIMENTAL DESIGN: Formalin-fixed, paraffin-embedded tissue microarrays were obtained from an adjuvant colon cancer trial (PETACC3) where patients were randomized to 5-fluorouracil/folinic acid with or without additional irinotecan.TOP1copy number status was analyzed by fluorescencein situhybridization (FISH) using aTOP1/CEN20 dual-probe combination.TOP1mRNA data were available from previous analyses. RESULTS: TOP1FISH and follow-up data were obtained from 534 patients.TOP1gain was identified in 27% using a single-probe enumeration strategy (≥4TOP1signals per cell) and in 31% when defined by aTOP1/CEN20 ratio ≥ 1.5. The effect of additional irinotecan was not dependent onTOP1FISH status.TOP1mRNA data were available from 580 patients with stage III disease. Benefit of irinotecan was restricted to patients characterized byTOP1mRNA expression ≥ third quartile (RFS: HRadjusted, 0.59;P= 0.09; OS: HRadjusted, 0.44;P= 0.03). The treatment byTOP1mRNA interaction was not statistically significant, but in exploratory multivariable fractional polynomial interaction analysis, increasingTOP1mRNA values appeared to be associated with increasing benefit of irinotecan. CONCLUSIONS: In contrast to theTOP1copy number, a trend was demonstrated for a predictive property ofTOP1mRNA expression. On the basis ofTOP1mRNA, it might be possible to identify a subgroup of patients where an irinotecan doublet is a clinically relevant option in the adjuvant setting of colon cancer.Clin Cancer Res; 22(7); 1621-31. ©2015 AACR.