Tuning the RNAPII elongation rate is required for optimal pre-mRNA splicing efficiency and fidelity

Splicing mainly occurs co-transcriptionally, suggesting that transcription and premRNA splicing could be synchronized. The nature of this phenomenon suggests that transcription elongation rate may influence splicing outcomes and, indeed, there is evidence for effects on alternative splicing in mammals. To elucidate potential effects of transcription rate on splicing efficiency and fidelity, splicing of nascent transcripts was investigated in fast and slow elongating RNA polymerase II (RNAPII) mutants in Saccharomyces cerevisiae. High kinetic resolution 4-thio Uracil labelling of nascent RNA reveals that fast RNAPII accumulates unspliced pre-mRNA that represents reduced co-transcriptional splicing. Conversely, low levels of unspliced pre-mRNA were detected in the slow mutant due to increased co-transcriptional splicing. The highly stable association of nascent transcripts with elongating RNAPII permits co-transcriptional splicing to be measured by analysis of transcripts that co-purify with RNAPII. Measuring co-precipitation of the spliced mRNA and excised intron that are associated with RNAPII demonstrates that splicing is mostly co-transcriptional with the slow mutant, and the fast mutant reduces co-transcriptional splicing. How elongation rate affects splicing fidelity in budding yeast and whether faster and slower transcription have the opposite effect on splicing fidelity as might be predicted by the kinetic coupling model is an open question. Using deep RNA sequencing, splicing fidelity was determined in yeast transcription elongation mutants. Results show that both fast and slow transcription reduce splicing fidelity mainly in ribosomal protein coding transcripts. Analysis reveals that splicing fidelity depends largely on intron length, secondary structure and splice site score. These analyses also provide new insights regarding the effect of altering transcription rate on selection of transcription start sites. Together, these results indicate that optimal splicing efficiency and fidelity require finely-tuned transcription speed

Global profiling of alternative splicing events and gene expression regulated by hnRNPH/F

In this study, we have investigated the global impact of heterogeneous nuclear Ribonuclear Protein (hnRNP) H/F-mediated regulation of splicing events and gene expression in oligodendrocytes. We have performed a genome-wide transcriptomic analysis at the gene and exon levels in Oli-neu cells treated with siRNA that targets hnRNPH/F compared to untreated cells using Affymetrix Exon Array. Gene expression levels and regulated exons were identified with the GenoSplice EASANA algorithm. Bioinformatics analyses were performed to determine the structural properties of G tracts that correlate with the function of hnRNPH/F as enhancers vs. repressors of exon inclusion. Different types of alternatively spliced events are regulated by hnRNPH/F. Intronic G tracts density, length and proximity to the 5\u27 splice site correlate with the hnRNPH/F enhancer function. Additionally, 6% of genes are differently expressed upon knock down of hnRNPH/F. Genes that regulate the transition of oligodendrocyte progenitor cells to oligodendrocytes are differentially expressed in hnRNPH/F depleted Oli-neu cells, resulting in a decrease of negative regulators and an increase of differentiation-inducing regulators. The changes were confirmed in developing oligodendrocytes in vivo. This is the first genome wide analysis of splicing events and gene expression regulated by hnRNPH/F in oligodendrocytes and the first report that hnRNPH/F regulate genes that are involved in the transition from oligodendrocyte progenitor cells to oligodendrocytes

Molecular Characterization of a Patient Presumed to Have Prader-Willi Syndrome

Prader-Willi syndrome (PWS) is caused by the loss of RNA expression from an imprinted region on chromosome 15 that includes SNRPN, SNORD115, and SNORD116. Currently, there are no mouse models that faithfully reflect the human phenotype and investigations rely on human post-mortem material. During molecular characterization of tissue deposited in a public brain bank from a patient diagnosed with Prader-Willi syndrome, we found RNA expression from SNRPN, SNORD115, and SNORD116 which does not support a genetic diagnosis of Prader-Willi syndrome. The patient was a female, Caucasian nursing home resident with history of morbid obesity (BMI 56.3) and mental retardation. She died at age of 56 from pulmonary embolism. SNORD115 and SNORD116 are unexpectedly stable in post mortem tissue and can be used for post-mortem diagnosis. Molecular characterization of PWS tissue donors can confirm the diagnosis and identify those patients that have been misdiagnosed

Global Profiling of Alternative Splicing Events and Gene Expression Regulated by hnRNPH/F

<div><p>In this study, we have investigated the global impact of heterogeneous nuclear Ribonuclear Protein (hnRNP) H/F-mediated regulation of splicing events and gene expression in oligodendrocytes. We have performed a genome-wide transcriptomic analysis at the gene and exon levels in Oli-neu cells treated with siRNA that targets hnRNPH/F compared to untreated cells using Affymetrix Exon Array. Gene expression levels and regulated exons were identified with the GenoSplice EASANA algorithm. Bioinformatics analyses were performed to determine the structural properties of G tracts that correlate with the function of hnRNPH/F as enhancers vs. repressors of exon inclusion. Different types of alternatively spliced events are regulated by hnRNPH/F. Intronic G tracts density, length and proximity to the 5′ splice site correlate with the hnRNPH/F enhancer function. Additionally, 6% of genes are differently expressed upon knock down of hnRNPH/F. Genes that regulate the transition of oligodendrocyte progenitor cells to oligodendrocytes are differentially expressed in hnRNPH/F depleted Oli-neu cells, resulting in a decrease of negative regulators and an increase of differentiation-inducing regulators. The changes were confirmed in developing oligodendrocytes <em>in vivo</em>. This is the first genome wide analysis of splicing events and gene expression regulated by hnRNPH/F in oligodendrocytes and the first report that hnRNPH/F regulate genes that are involved in the transition from oligodendrocyte progenitor cells to oligodendrocytes.</p> </div

Molecular Characterization of a Patient Presumed to Have Prader-Willi Syndrome

Prader-Willi syndrome (PWS) is caused by the loss of RNA expression from an imprinted region on chromosome 15 that includes SNRPN, SNORD115, and SNORD116. Currently, there are no mouse models that faithfully reflect the human phenotype and investigations rely on human post-mortem material. During molecular characterization of tissue deposited in a public brain bank from a patient diagnosed with Prader-Willi syndrome, we found RNA expression from SNRPN, SNORD115, and SNORD116 which does not support a genetic diagnosis of Prader-Willi syndrome. The patient was a female, Caucasian nursing home resident with history of morbid obesity (BMI 56.3) and mental retardation. She died at age of 56 from pulmonary embolism. SNORD115 and SNORD116 are unexpectedly stable in post mortem tissue and can be used for post-mortem diagnosis. Molecular characterization of PWS tissue donors can confirm the diagnosis and identify those patients that have been misdiagnosed

Genes are differentially regulated at the transcriptional levels.

<p><b>A.</b> Representation of significant pathways whose genes are affected by knock down of hnRNPH/F. Thirty one Kegg pathways were significantly affected. Genes are either up- or down-regulated. <b>B.</b> Changes in gene expression were verified by Real Time RT-PCR in Oli-neu cells depleted of hnRNPH and F. Bar graphs represent the mean±SD of transcript levels of the indicated genes quantitated by Real Time RT-PCR in mock siRNA treated (Mock) and siF/H treated Oli-neu cells (n = 3). Oli-neu cells were treated with siF/H and harvested after 72 hrs in culture for Real Time RT-PCR analysis. The data are expressed as percent change of the treated vs. mock cells, the latter is set at the value of 1. ns = non significant, *p = 0.05. In parenthesis next to the gene name is shown the fold change in the microarrays.</p

Changes in exon inclusion induced by silencing hnRNPH and F individually vs. both simultaneously.

<p>Representative RT-PCR (n = 2) of the products derived from 5′ASEs in Oli-neu cells treated with siRNAs that target hnRNPH (siH), hnRNPF (siF) or both (siF/H). We selected ASEs that were shown to have a statistically significant change in exon inclusion by RT-PCR. Mock are control untreated Oli-neu cells. Each ASE is labeled with the gene ID number and the ae is shown (also refer to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051266#pone-0051266-g001" target="_blank">Figure 1E</a>). The number shown below each lane represents the fold change in exon inclusion compared to the mock treated cells set at the value of 1. The PLP/DM20 splicing event shows the synergistic effect of hnRNPH/F knock down.</p

Genome wide analysis of alternative spliced events (ASEs) regulated by hnRNPH/F.

<p>Oli-neu cells were treated with an siRNA, siF/H that targets both hnRNPH and F <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051266#pone.0051266-Wang3" target="_blank">[12]</a>. RNA was used to generate probes to hybridize with the Affymetrix Mouse exon 1.0ST array featuring ∼ 1 million exon clusters and 1.4 million probe sets. We have analyzed splicing and transcript levels using the EASANA® from GenoSplice technology (<a href="http://www.genosplice.com" target="_blank">www.genosplice.com</a>). <b>A.</b> RT-PCR amplification of the endogenous PLP and DM20 transcripts. Schematic of the PCR products is shown. siF/H treatment induces a two-fold increase in the inclusion of exon 3B. Percent inclusion of exon 3B is shown. <b>B.</b> Western blot analysis of hnRNPH and F expression. More than 70% reduction of hnRNPH/F is induced by the siF/H treatment. hnRNPA1 is used as loading control. <b>C.</b> Pie chart showing the ASEs regulated by hnRNPH/F. Splicing of 252 exons was differentially regulated by knock down of hnRNPH/F. The types of spliced events are shown. <b>D.</b> Pie charts show the relative abundance of included (hnRNPH/F-repressed) and excluded (hnRNPH/F-activated) exons. For four of the six categories of alternative spliced events a greater number of exons are excluded (i.e. hnRNPH/F-activated) by depletion of hnRNPH/F. <b>E.</b> RT-PCR of alternative 5′ splice sites. Representative RT-PCR of 5′ASEs that were examined by semiquantitative RT-PCR analysis in siF/H treated vs. untreated Oli-neu cells (mock) (n = 3). Each ASE is labeled with the gene ID and the alternative spliced exon (ae) is shown. The ASEs that were validated by RT-PCR are shown in bold. The others, not bolded, demonstrated a change that was in the opposite direction of that detected in the arrays. Bar graphs represent the percent change of the exon inclusion ± SD in the siF/H treated cells vs. untreated cells set at 1 (n = 3). *≤0.05 and **≤0.01.</p