RNA-binding protein CPEB1 remodels host and viral RNA landscapes.

Host and virus interactions occurring at the post-transcriptional level are critical for infection but remain poorly understood. Here, we performed comprehensive transcriptome-wide analyses revealing that human cytomegalovirus (HCMV) infection results in widespread alternative splicing (AS), shortening of 3' untranslated regions (3' UTRs) and lengthening of poly(A)-tails in host gene transcripts. We found that the host RNA-binding protein CPEB1 was highly induced after infection, and ectopic expression of CPEB1 in noninfected cells recapitulated infection-related post-transcriptional changes. CPEB1 was also required for poly(A)-tail lengthening of viral RNAs important for productive infection. Strikingly, depletion of CPEB1 reversed infection-related cytopathology and post-transcriptional changes, and decreased productive HCMV titers. Host RNA processing was also altered in herpes simplex virus-2 (HSV-2)-infected cells, thereby indicating that this phenomenon might be a common occurrence during herpesvirus infections. We anticipate that our work may serve as a starting point for therapeutic targeting of host RNA-binding proteins in herpesvirus infections

Distinct and shared functions of ALS-associated proteins TDP-43, FUS and TAF15 revealed by multisystem analyses

The RNA-binding protein (RBP) TAF15 is implicated in amyotrophic lateral sclerosis (ALS). To compare TAF15 function to that of two ALS-associated RBPs, FUS and TDP-43, we integrate CLIP-seq and RNA Bind-N-Seq technologies, and show that TAF15 binds to ∼4,900 RNAs enriched for GGUA motifs in adult mouse brains. TAF15 and FUS exhibit similar binding patterns in introns, are enriched in 3′ untranslated regions and alter genes distinct from TDP-43. However, unlike FUS and TDP-43, TAF15 has a minimal role in alternative splicing. In human neural progenitors, TAF15 and FUS affect turnover of their RNA targets. In human stem cell-derived motor neurons, the RNA profile associated with concomitant loss of both TAF15 and FUS resembles that observed in the presence of the ALS-associated mutation FUS R521G, but contrasts with late-stage sporadic ALS patients. Taken together, our findings reveal convergent and divergent roles for FUS, TAF15 and TDP-43 in RNA metabolism.National Institutes of Health (U.S.) (Grant HG007005

Comprehensive discovery and analysis of RNA binding protein-dependent post-transcriptional events in mammalian systems /

Nascent transcripts produced by RNA polymerase II in eukaryotic cells are subject to extensive processing prior to the generation of a functional messenger RNA (mRNA). These RNAs are generally found coated with RNA binding proteins (RBPs), which act in concert to regulate RNA processing events, including splicing, polyadenylation, and RNA stability. The aggregate effect of various RBPs on a given RNA transcript eventually dictates its fate, a phenomenon referred to as the RNP code. The precise control of RNA processing by RBPs is incredibly important for cellular homeostasis, defects of which lead to numerous accounts of human genetic diseases in many tissues. With the emergence of genome-wide methods for detecting direct binding of RBPs on target RNAs, coupled with technologies to measure changes in various aspects of RNA processing, global rules and insights for individual RBPs have been revealed. However, few studies have combined regulatory changes for more than one RBP to better understand their combinatorial effects on RNA targets. Additionally, despite the importance of RBPs, we still do not have the complete repertoire of which proteins bind to RNA, and which of these bind simultaneously with other RBPs to constitute the "RBP-RNA interactome". Here, I conduct genome-wide RNA processing analyses in mammalian cells, integrating regulation and binding information for multiple RBPs, including disease- related RBPs, the highly abundant heterogeneous nuclear ribonucleoparticle (hnRNP) proteins, and many previously uncharacterized RBPs. This battery of computational and experimental assays provides insight into the unique roles of hundreds of individual RBPs, as well as the extent of coordinated regulation between RBPs. I also describe a systematic approach to identifying the proteins that interact with RNA simultaneously with hnRNP proteins. Not limited to only mRNA-bound proteins, my strategy identifies thousands of hnRNP protein interactors, including putative novel proteins that interact with mRNA and pre-mRNA, and have biochemical and statistical attributes of known RBPs. My findings expand the repertoire of RNA-interacting RBPs and provide a resource for the study of human simultaneous RBP-RBP interactomes. This comprehensive analysis of RBPs investigates their specific roles in the regulation of RNA processing yielding interesting findings for the RNA biology field and insights into how misregulation can impact human diseas

An Efficient Method for Identifying Gene Fusions by Targeted RNA Sequencing from Fresh Frozen and FFPE Samples

<div><p>Fusion genes are known to be key drivers of tumor growth in several types of cancer. Traditionally, detecting fusion genes has been a difficult task based on fluorescent <i>in situ</i> hybridization to detect chromosomal abnormalities. More recently, RNA sequencing has enabled an increased pace of fusion gene identification. However, RNA-Seq is inefficient for the identification of fusion genes due to the high number of sequencing reads needed to detect the small number of fusion transcripts present in cells of interest. Here we describe a method, Single Primer Enrichment Technology (SPET), for targeted RNA sequencing that is customizable to any target genes, is simple to use, and efficiently detects gene fusions. Using SPET to target 5701 exons of 401 known cancer fusion genes for sequencing, we were able to identify known and previously unreported gene fusions from both fresh-frozen and formalin-fixed paraffin-embedded (FFPE) tissue RNA in both normal tissue and cancer cells.</p></div

EIF4E3-FOXP1 fusion transcript.

<p>(A) Structure of the fusion transcript based on UCSC genome browser tracks. Red arrow indicates the in frame fusion of EIF4E3 exon7 to FOXP1 exon 4. The Sanger sequencing trace below indicates the sequence of the fusion point, while the blue arrow over the sanger sequence trace indicates the 3’ end of the targeting probe. (B) RT-PCR result using PCR primers indicated described in S2 for detecting this fusion transcript. Lane 1: RT-PCR product showing the correct 209bp size, Lane 2: 50bp ladder.</p

HCC1937 Breast ductal carcinoma RNA.

<p>(A) FFPE RNA Bioanalyzer trace. (B) Bioanalyzer trace of sequencing library derived from 100 ng of RNA input shown in A. (C) Sequencing metrics for targeted RNA show that FFPE RNA is efficiently targeted.</p

Crystal Structure of a Cytochrome P450 2B6 Genetic Variant in Complex with the Inhibitor 4-(4-Chlorophenyl)imidazole at 2.0-Å Resolution

The structure of the K262R genetic variant of human cytochrome P450 2B6 in complex with the inhibitor 4-(4-chlorophenyl)imidazole (4-CPI) has been determined using X-ray crystallography to 2.0-Å resolution. Production of diffraction quality crystals was enabled through a combination of protein engineering, chaperone coexpression, modifications to the purification protocol, and the use of unique facial amphiphiles during crystallization. The 2B6-4-CPI complex is virtually identical to the rabbit 2B4 structure bound to the same inhibitor with respect to the arrangement of secondary structural elements and the placement of active site residues. The structure supports prior P450 2B6 homology models based on other mammalian cytochromes P450 and is consistent with the limited site-directed mutagenesis studies on 2B6 and extensive studies on P450 2B4 and 2B1. Although the K262R genetic variant shows unaltered binding of 4-CPI, altered binding affinity, kinetics, and/or product profiles have been previously shown with several other ligands. On the basis of new P450 2B6 crystal structure and previous 2B4 structures, substitutions at residue 262 affect a hydrogen-bonding network connecting the G and H helices, where subtle differences could be transduced to the active site. Docking experiments indicate that the closed protein conformation allows smaller ligands such as ticlopidine to bind to the 2B6 active site in the expected orientation. However, it is unknown whether 2B6 undergoes structural reorganization to accommodate bulkier molecules, as previously inferred from multiple P450 2B4 crystal structures

Integrative Genome-wide Analysis Reveals Cooperative Regulation of Alternative Splicing by hnRNP Proteins

Understanding how RNA binding proteins control the splicing code is fundamental to human biology and disease. Here, we present a comprehensive study to elucidate how heterogeneous nuclear ribonucleoparticle (hnRNP) proteins, among the most abundant RNA binding proteins, coordinate to regulate alternative pre-mRNA splicing (AS) in human cells. Using splicing-sensitive microarrays, crosslinking and immunoprecipitation coupled with high-throughput sequencing (CLIP-seq), and cDNA sequencing, we find that more than half of all AS events are regulated by multiple hnRNP proteins and that some combinations of hnRNP proteins exhibit significant synergy, whereas others act antagonistically. Our analyses reveal position-dependent RNA splicing maps, in vivo consensus binding sites, a surprising level of cross- and autoregulation among hnRNP proteins, and the coordinated regulation by hnRNP proteins of dozens of other RNA binding proteins and genes associated with cancer. Our findings define an unprecedented degree of complexity and compensatory relationships among hnRNP proteins and their splicing targets that likely confer robustness to cells

ALS-linked TDP-43 mutations produce aberrant RNA splicing and adult-onset motor neuron disease without aggregation or loss of nuclear TDP-43

Transactivating response region DNA binding protein (TDP-43) is the major protein component of ubiquitinated inclusions found in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD) with ubiquitinated inclusions. Two ALS-causing mutants (TDP-43(Q331K) and TDP-43(M337V)), but not wild-type human TDP-43, are shown here to provoke age-dependent, mutant-dependent, progressive motor axon degeneration and motor neuron death when expressed in mice at levels and in a cell type-selective pattern similar to endogenous TDP-43. Mutant TDP-43-dependent degeneration of lower motor neurons occurs without: (i) loss of TDP-43 from the corresponding nuclei, (ii) accumulation of TDP-43 aggregates, and (iii) accumulation of insoluble TDP-43. Computational analysis using splicing-sensitive microarrays demonstrates alterations of endogenous TDP-43–dependent alternative splicing events conferred by both human wild-type and mutant TDP-43(Q331K), but with high levels of mutant TDP-43 preferentially enhancing exon exclusion of some target pre-mRNAs affecting genes involved in neurological transmission and function. Comparison with splicing alterations following TDP-43 depletion demonstrates that TDP-43(Q331K) enhances normal TDP-43 splicing function for some RNA targets but loss-of-function for others. Thus, adult-onset motor neuron disease does not require aggregation or loss of nuclear TDP-43, with ALS-linked mutants producing loss and gain of splicing function of selected RNA targets at an early disease stage