CLIP and complementary methods

RNA molecules start assembling into ribonucleoprotein (RNP) complexes during transcription. Dynamic RNP assembly, largely directed by cis-acting elements on the RNA, coordinates all processes in which the RNA is involved. To identify the sites bound by a specific RNA-binding protein on endogenous RNAs, cross-linking and immunoprecipitation (CLIP) and complementary, proximity-based methods have been developed. In this Primer, we discuss the main variants of these protein-centric methods and the strategies for their optimization and quality assessment, as well as RNA-centric methods that identify the protein partners of a specific RNA. We summarize the main challenges of computational CLIP data analysis, how to handle various sources of background and how to identify functionally relevant binding regions. We outline the various applications of CLIP and available databases for data sharing. We discuss the prospect of integrating data obtained by CLIP with complementary methods to gain a comprehensive view of RNP assembly and remodelling, unravel the spatial and temporal dynamics of RNPs in specific cell types and subcellular compartments and understand how defects in RNPs can lead to disease. Finally, we present open questions in the field and give directions for further development and applications

Kepler Cycle 1 Observations of Low Mass Stars: New Eclipsing Binaries, Single Star Rotation Rates, and the Nature and Frequency of Starspots

We have analyzed Kepler light curves for 849 stars with T_eff < 5200 K from our Cycle 1 Guest Observer program. We identify six new eclipsing binaries, one of which has an orbital period of 29.91 d, and two of which are probably W UMa variables. In addition, we identify a candidate "warm Jupiter" exoplanet. We further examine a subset of 670 sources for variability. Of these objects, 265 stars clearly show periodic variability that we assign to rotation of the low-mass star. At the photometric precision level provided by Kepler, 251 of our objects showed no evidence for variability. We were unable to determine periods for 154 variable objects. We find that 79% of stars with T_eff < 5200 K are variable. The rotation periods we derive for the periodic variables span the range 0.31 < P_rot < 126.5 d. A considerable number of stars with rotation periods similar to the solar value show activity levels that are 100 times higher than the Sun. This is consistent with results for solar-like field stars. As has been found in previous studies, stars with shorter rotation periods generally exhibit larger modulations. This trend flattens beyond P_rot = 25 d, demonstrating that even long period binaries may still have components with high levels of activity and investigating whether the masses and radii of the stellar components in these systems are consistent with stellar models could remain problematic. Surprisingly, our modeling of the light curves suggests that the active regions on these cool stars are either preferentially located near the rotational poles, or that there are two spot groups located at lower latitudes, but in opposing hemispheres.Comment: 48 pages, 11 figure

Low-Mass Eclipsing Binaries in the Initial Kepler Data Release

We identify 231 objects in the newly released Cycle 0 dataset from the Kepler Mission as double-eclipse, detached eclipsing binary systems with Teff < 5500 K and orbital periods shorter than ~32 days. We model each light curve using the JKTEBOP code with a genetic algorithm to obtain precise values for each system. We identify 95 new systems with both components below 1.0 M_sun and eclipses of at least 0.1 magnitudes, suitable for ground-based follow-up. Of these, 14 have periods less than 1.0 day, 52 have periods between 1.0 and 10.0 days, and 29 have periods greater than 10.0 days. This new sample of main-sequence, low-mass, double-eclipse, detached eclipsing binary candidates more than doubles the number of previously known systems, and extends the sample into the completely heretofore unexplored P > 10.0 day period regime. We find preliminary evidence from these systems that the radii of low-mass stars in binary systems decrease with period. This supports the theory that binary spin-up is the primary cause of inflated radii in low-mass binary systems, although a full analysis of each system with radial-velocity and multi-color light curves is needed to fully explore this hypothesis. As well, we present 7 new transiting planet candidates that do not appear among the recently released list of 706 candidates by the Kepler team, nor in the Kepler False Positive Catalog, along with several other new and interesting systems. We also present novel techniques for the identification, period analysis, and modeling of eclipsing binaries.Comment: 22 pages in emulateapj format. 9 figures, 4 tables, 2 appendices. Accepted to AJ. Includes a significant addition of new material since last arXiv submission and an updated method for estimating masses and radi

The paralogues MAGOH and MAGOHB are oncogenic factors in high-grade gliomas and safeguard the splicing of cell division and cell cycle genes

The exon junction complex (EJC) plays key roles throughout the lifespan of RNA and is particularly relevant in the nervous system. We investigated the roles of two EJC members, the paralogs MAGOH and MAGOHB, with respect to brain tumour development. High MAGOH/MAGOHB expression was observed in 14 tumour types; glioblastoma (GBM) showed the greatest difference compared to normal tissue. Increased MAGOH/MAGOHB expression was associated with poor prognosis in glioma patients, while knockdown of MAGOH/MAGOHB affected different cancer phenotypes. Reduced MAGOH/MAGOHB expression in GBM cells caused alterations in the splicing profile, including re-splicing and skipping of multiple exons. The binding profiles of EJC proteins indicated that exons affected by MAGOH/MAGOHB knockdown accumulated fewer complexes on average, providing a possible explanation for their sensitivity to MAGOH/MAGOHB knockdown. Transcripts (genes) showing alterations in the splicing profile are mainly implicated in cell division, cell cycle, splicing, and translation. We propose that high MAGOH/MAGOHB levels are required to safeguard the splicing of genes in high demand in scenarios requiring increased cell proliferation (brain development and GBM growth), ensuring efficient cell division, cell cycle regulation, and gene expression (splicing and translation). Since differentiated neuronal cells do not require increased MAGOH/MAGOHB expression, targeting these paralogs is a potential option for treating GBM

iCLIP Predicts the Dual Splicing Effects of TIA-RNA Interactions

Transcriptome-wide analysis of protein-RNA interactions predicts the dual splicing effects of TIA proteins, showing that their local enhancing function is associated with diverse distal splicing silencing effects

Enrichment analysis of Alu elements with different spatial chromatin proximity in the human genome

Transposable elements (TEs) have no longer been totally considered as “junk DNA” for quite a time since the continual discoveries of their multifunctional roles in eukaryote genomes. As one of the most important and abundant TEs that still active in human genome, Alu, a SINE family, has demonstrated its indispensable regulatory functions at sequence level, but its spatial roles are still unclear. Technologies based on 3C(chromosomeconformation capture) have revealed the mysterious three-dimensional structure of chromatin, and make it possible to study the distal chromatin interaction in the genome. To find the role TE playing in distal regulation in human genome, we compiled the new released Hi-C data, TE annotation, histone marker annotations, and the genome-wide methylation data to operate correlation analysis, and found that the density of Alu elements showed a strong positive correlation with the level of chromatin interactions (hESC: r=0.9, P<2.2×1016; IMR90 fibroblasts: r = 0.94, P < 2.2 × 1016) and also have a significant positive correlation withsomeremote functional DNA elements like enhancers and promoters (Enhancer: hESC: r=0.997, P=2.3×10−4; IMR90: r=0.934, P=2×10−2; Promoter: hESC: r = 0.995, P = 3.8 × 10−4; IMR90: r = 0.996, P = 3.2 × 10−4). Further investigation involving GC content and methylation status showed the GC content of Alu covered sequences shared a similar pattern with that of the overall sequence, suggesting that Alu elements also function as the GC nucleotide and CpG site provider. In all, our results suggest that the Alu elements may act as an alternative parameter to evaluate the Hi-C data, which is confirmed by the correlation analysis of Alu elements and histone markers. Moreover, the GC-rich Alu sequence can bring high GC content and methylation flexibility to the regions with more distal chromatin contact, regulating the transcription of tissue-specific genes

Aberrant methylation of tRNAs links cellular stress to neuro-developmental disorders.

Mutations in the cytosine-5 RNA methyltransferase NSun2 cause microcephaly and other neurological abnormalities in mice and human. How post-transcriptional methylation contributes to the human disease is currently unknown. By comparing gene expression data with global cytosine-5 RNA methylomes in patient fibroblasts and NSun2-deficient mice, we find that loss of cytosine-5 RNA methylation increases the angiogenin-mediated endonucleolytic cleavage of transfer RNAs (tRNA) leading to an accumulation of 5' tRNA-derived small RNA fragments. Accumulation of 5' tRNA fragments in the absence of NSun2 reduces protein translation rates and activates stress pathways leading to reduced cell size and increased apoptosis of cortical, hippocampal and striatal neurons. Mechanistically, we demonstrate that angiogenin binds with higher affinity to tRNAs lacking site-specific NSun2-mediated methylation and that the presence of 5' tRNA fragments is sufficient and required to trigger cellular stress responses. Furthermore, the enhanced sensitivity of NSun2-deficient brains to oxidative stress can be rescued through inhibition of angiogenin during embryogenesis. In conclusion, failure in NSun2-mediated tRNA methylation contributes to human diseases via stress-induced RNA cleavage

P-TEFb activation by RBM7 shapes a pro-survival transcriptional response to genotoxic stress

Cellular DNA damage response (DDR) involves dramatic transcriptional alterations, the mechanisms of which remain ill-defined. Given the centrality of RNA polymerase II (Pol II) promoter-proximal pause release in transcriptional control, we evaluated its importance in DDR. Here we show that following genotoxic stress, the RNA-binding motif protein 7 (RBM7) stimulates Pol II elongation and promotes cell viability by activating the positive transcription elongation factor b (P-TEFb). This is mediated by genotoxic stress-enhanced binding of RBM7 to 7SK snRNA (7SK), the scaffold of the 7SK small nuclear ribonucleoprotein (7SK snRNP) which inhibits P-TEFb. In turn, P-TEFb relocates from 7SK snRNP to chromatin to induce transcription of short units including key DDR genes and multiple classes of non-coding RNAs. Critically, interfering with RBM7 or P-TEFb provokes cellular hypersensitivity to DNA damage-inducing agents through activation of apoptotic program. By alleviating the inhibition of P-TEFb, RBM7 thus facilitates Pol II elongation to enable a pro-survival transcriptional response that is crucial for cell fate upon genotoxic insult. Our work uncovers a new paradigm in stress-dependent control of Pol II pause release, and offers the promise for designing novel anti-cancer interventions using RBM7 and P-TEFb antagonists in combination with DNA-damaging chemotherapeutics