Oriental beat album artwork3/20/2023 Interestingly, when using the photoactivatable 4-thiouridine (4SU)-based crosslinking in combination with iCLIP, the motifs were more highly enriched at cDNA-starts than at T-to-C transitions. Moreover, we identified RNA motifs that are commonly associated with crosslink sites and found them most highly enriched at cDNA deletions in CLIP, and cDNA-starts in iCLIP, eCLIP and irCLIP, even if non-coinciding cDNA-starts are prevalent. We first examined the position and prevalence of crosslink-induced mutations to confirm previous findings, showing that such mutations are generally >5-fold less common within iCLIP than CLIP cDNAs, regardless of the presence of non-coinciding cDNA-starts. To understand if cDNA-starts can be used to assign RNA-binding sites, we further analysed the iCLIP data with high frequency of non-coinciding cDNA-starts. The study concluded that the use of cDNA-starts may not be appropriate in iCLIP whenever non-coinciding cDNA-starts are prevalent. ![]() The non-coinciding cDNA-starts in eIF4A3 iCLIP data produced by the previous study were shifted upstream of this expected region and it was proposed that the presence of non-coinciding cDNA-starts might be related to this shift. However, further studies showed that the sequence and structure of a nascent messenger RNA (mRNA) can shift EJC deposition as far as 10 nt away from this expected site. In vitro biochemical experiments with several splicing substrates demonstrated that the site of EJC deposition is normally expected at nucleotides −20 to −24 upstream of the exon-exon junction (–24.–20 nt). eIF4A3 is a component of the exon junction complex (EJC). Here, we focused on experiments produced for polypyrimidine tract binding protein 1 (PTBP1), eukaryotic initiation factor 4A-III (eIF4A3) and the splicing factor U2 auxiliary factor 65 kDa subunit (U2AF2), which represent examples of non-coinciding or coinciding cDNA-starts in introns or exons. However, a recent study observed that the starts of long and short iCLIP cDNAs often map to different genomic positions for several RBPs, which leads to non-coinciding cDNA-starts. The computational methods that use cDNA-starts to assign RNA-binding sites have been developed along with iCLIP. Therefore, understanding the proportion and characteristics of truncated cDNAs in these protocols is essential. Recently, further variants were developed that also amplify truncated cDNAs, including BrdU-CLIP, eCLIP and irCLIP. Even though iCLIP amplifies both truncated and readthrough cDNAs, computational comparisons of CLIP and iCLIP cDNAs estimated that over 80% of iCLIP cDNAs truncate at the crosslink sites of most RBPs. The cDNA-starts of these truncated cDNAs identify the nucleotide just downstream of the crosslinked peptide. Therefore, individual-nucleotide resolution CLIP (iCLIP) was also developed to exploit the ‘truncated cDNAs’. The CLIP protocol prepares the cDNA library in a way that requires the reverse transcriptase to read through this peptide, thereby generating only ‘readthrough cDNAs’. This is followed by reverse transcription, during which the bound peptide can lead to truncation of complementary DNAs (cDNA) at the crosslink site. ![]() During the CLIP protocol, crosslinked protein–RNA complexes are purified and the RNA fragments are released by digesting the protein, resulting in RNAs with a covalently bound peptide at the crosslink site. To understand the mechanisms of their action, it is essential to identify the endogenous sites of protein–RNA interactions, which has been aided by the development of ultraviolet (UV) crosslinking and immunoprecipitation (CLIP). Created by Hilary Paynter, the sleeve couldn’t be a more visually perfect representation of the music within.RNA-binding proteins (RBPs) play crucial roles in all aspects of post-transcriptional gene regulation. After the band’s creative director, Edwin Burdis, heard the album, he told the group he imagined its artwork would feature wood engravings – and so it does. Where their previous three albums featured the band on the cover, Ribbon Around The Bomb’s sleeve fits a different brief, emerging as a visually striking classic among the best album covers of 2022. Blossoms threw a curveball into the mix with this one, and that decision very much extended to the artwork. A complex amalgamation of influences including wedding cheese, Simon And Garfunkel-style acoustics and a lot of theatrics, the record runs with creativity and ensures that each track is a personality-filled nugget of its own. Wiping the slate clean, Blossoms stepped away from their all-encompassing indie-pop jangles and headed into new territory in every sense on their 2022 album, Ribbon Around The Bomb.
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