1/29/2024 0 Comments Iclip biotin![]() ![]() Our results uncover the multifaceted role of DDX21 in multiple steps of ribosome biogenesis, and provide evidence implicating a mammalian RNA helicase in RNA modification and Pol II elongation control. Promoter-bound DDX21 facilitates the release of the positive transcription elongation factor b (P-TEFb) from the 7SK snRNP in a manner that is dependent on its helicase activity, thereby promoting transcription of its target genes. In the nucleoplasm, DDX21 binds 7SK RNA and, as a component of the 7SK small nuclear ribonucleoprotein (snRNP) complex, is recruited to the promoters of Pol II-transcribed genes encoding ribosomal proteins and snoRNAs. In the nucleolus, DDX21 occupies the transcribed rDNA locus, directly contacts both rRNA and snoRNAs, and promotes rRNA transcription, processing and modification. Although broad, these molecular interactions, both at the chromatin and RNA level, exhibit remarkable specificity for the regulation of ribosomal genes. We demonstrate that DDX21 widely associates with Pol I- and Pol II-transcribed genes and with diverse species of RNA, most prominently with non-coding RNAs involved in the formation of ribo-nucleoprotein complexes, including ribosomal RNA, small nucleolar RNAs (snoRNAs) and 7SK RNA. Here we show that the DEAD-box RNA helicase DDX21 can sense the transcriptional status of both RNA polymerase (Pol) I and II to control multiple steps of ribosome biogenesis in human cells. ![]() High-throughput sequencing generates reads where the barcode sequences are immediately before the last nucleotide of the input cDNA (step 16).DEAD-box RNA helicases are vital for the regulation of various aspects of the RNA life cycle 1, but the molecular underpinnings of their involvement, particularly in mammalian cells, remain poorly understood. Linearization generates templates for PCR amplification (steps 14 and 15). Size selection using Urea-PAGE removes the RT primer prior to circularization of the cDNA (steps 11–13). Reverse transcription truncates at the remaining polypeptide and introduces two cleavable adapter regions and a barcode (step 10). Complexes are recovered from the membrane using Proteinase K (step 9). RBP–RNA complexes are purified from free RNA using SDS-PAGE and wet membrane transfer (step 8). 元 linker adapter ligation to the 3′ end allows for sequence-specific priming of reverse transcription and the 5′ is radioactively labeled (steps 6 and 7). Potential RBP–RNA complexes are then purified together (steps 2–5). RNA binding protein (RBP) and RNA are covalently bound in vivo using UV radiation (step 1). Immunoprecipitation RBP–RNA complex RNA binding proteins UV crosslinking immunoprecipitation iCLIP. Here, we describe the detailed procedure of iCLIP. This method, individual nucleotide resolution CLIP (iCLIP) circularizes cDNA to capture the truncated cDNA and also increases the efficiency of ligating sequencing adapters to the library. This is harnessed by one variant of CLIP methods to identify crosslinking sites at a nucleotide resolution. As a result, reverse transcriptase may not read through the crosslink sites, and produce cDNA ending at the crosslinked nucleotide. Meanwhile, one or more amino acids of a crosslinked RBP can remain attached to its bound RNA due to incomplete digestion of the protein. UV irradiation can result in cDNA truncations and/or mutations at the crosslink sites, which complicates the alignment of the sequencing library to the reference genome and the identification of the crosslinking sites. Reverse transcription of the RNA followed by high-throughput sequencing of the cDNA library is now often used to identify protein bound RNA on a genome-wide scale. The RBP is protease digested to allow purification of the bound RNA. After stringent washing and gel separation the RBP-RNA complex is excised. After partial RNase digestion, antibodies specific to an RNA binding protein (RBP) or a protein-epitope tag is then used to immunoprecipitate the protein-RNA complexes. Whole cells or tissues are ultraviolet irradiated to generate a covalent bond between RNA and proteins that are in close contact. UV crosslinking immunoprecipitation (CLIP) is an increasingly popular technique to study protein-RNA interactions in tissues and cells. ![]()
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