Identification of protein binding sites on U3 snoRNA and pre-rRNA by UV cross-linking and high-throughput analysis of cDNAs

Abstract

The U3 small nucleolar ribonucleoprotein (snoRNP) plays an essential role in ribosome biogenesis but, like many RNA-protein complexes, its architecture is poorly understood. To address this problem, binding sites for the snoRNP proteins Nop1, Nop56, Nop58, and Rrp9 were mapped by UV cross-linking and analysis of cDNAs. Cross-linked protein-RNA complexes were purified under highly-denaturing conditions, ensuring that only direct interactions were detected. Recovered RNA fragments were amplified after linker ligation and cDNA synthesis. Cross-linking was successfully performed either in vitro on purified complexes or in vivo in living cells. Cross-linking sites were precisely mapped either by Sanger sequencing of multiple cloned fragments or direct, high-throughput Solexa sequencing. Analysis of RNAs associated with the snoRNP proteins revealed remarkably high signal-to-noise ratios and identified specific binding sites for each of these proteins on the U3 RNA. The results were consistent with previous data, demonstrating the reliability of the method, but also provided insights into the architecture of the U3 snoRNP. The snoRNP proteins were also cross-linked to pre-rRNA fragments, with preferential association at known sites of box C/D snoRNA function. This finding demonstrates that the snoRNP proteins directly contact the pre-rRNA substrate, suggesting roles in snoRNA recruitment. The techniques reported here should be widely applicable to analyses of RNA-protein interactions.ribosome synthesis ͉ RNA modification ͉ RNA processing ͉ RNP structure ͉ yeast P roteomic approaches have identified many factors involved in ribosome synthesis in yeast, but we still lack detailed understanding of the architecture of the preribosomes and small nucleolar ribonucleoprotein (snoRNP) complexes that are required for their maturation. Several methods have been described that allow identification of protein-RNA interaction sites in native particles. RNA immunoprecipitation uses formaldehyde to cross-link RNA to proteins (1, 2). Caveats of this method are that formaldehyde also cross-links proteins to proteins and the immunoprecipitation step is performed under semidenaturing conditions, so a positive result does not demonstrate direct RNA-protein interaction, and the spatial resolution of the technique is low. Moreover, formaldehyde and other chemical cross-linkers may not enter the cores of large complexes. This problem can be avoided by cross-linking proteins and RNA with UV light, and several techniques have been reported (3-7).UV-induced protein-RNA cross-links can be detected by primer extension analysis on the RNA and by MALDI-MS on the protein (4). These approaches can detect cross-links on both protein and RNA but primer extension mapping on long RNAs is not practical without prior knowledge of the approximate cross-linking site and MS analyses require up to 50 pmol of RNP (5). The cross-linking and immunoprecipitation (CLIP) method identified protein-RNA interaction sites in mammalian cells by cloning of the covalentlya...

Murine cytomegalovirus encodes a miR-27 inhibitor disguised as a target

“…3). In CRAC and related techniques, an increased rate of microdeletions is seen in the sequence data at the site of protein crosslinking (19,(23)(24)(25)(26). This is attributable to errors introduced by reverse transcriptase when bypassing the crosslinked site and can be used to pinpoint the precise proteinbinding site.…”

“…NIH 3T3 cells (1.2 × 10 7 cells per sample, distributed in four 15-cm dishes) were transfected with 60 μg of pcDNA3-PTH-Ago2 plasmid in 0.3% Lipofectamine 2000 (Invitrogen) and 20 h posttransfection infected with MCMV (Smith strain) at an MOI of 5. At 8 h postinfection, cells were washed with PBS, immediately UV-irradiated at 254 nm (400 mJ/cm 2 ; Uvitec), and the CRAC pull-down technique was performed as described previously (19), with modifications detailed in SI Materials and Methods. For construction of the miR-27 sensor, three miR-27-binding sites spaced 11 nt apart were cloned behind the 3′UTR of Renilla luciferase in the psiCHECK2 vector (Promega) as in Ref.…”

“…miRNAs function within the RNA-induced silencing complex (RISC), the effectors of which are Argonaute (Ago) proteins. We adapted the UV-crosslinking and analysis of cDNA (CRAC) technique (19) to identify RNA sequences bound to the Ago2 protein. Living cells expressing tagged Ago2 were UV-irradiated at 254 nm, and RNA was purified and reverse transcribed, and the cDNAs were sequenced on a Solexa GAII.…”

“…Cells were crosslinked and lysed, and the RNA-protein complexes were purified as described (19), with modifications detailed in SI Materials and Methods. RNA classes identified in uninfected and infected cells are indicated in Table S1.…”

Analysis of protein–RNA interactions in CRISPR proteins and effector complexes by UV-induced cross-linking and mass spectrometry

“…This approach is complementary to other UV-induced cross-linking approaches such as PAR-CLIP [59] and CRAC [60], in which next-generation sequencing techniques are applied in order to identify the nucleotides cross-linked to the proteins of interest. Combining of both these approaches in future studies promises an unprecedented insight into RBP biology at both the protein and the RNA level.…”