RNA 결합 단백질(RBP)은 서열과 구조적 모티프의 인식을 통해 RNA에 결합하여 세포 유형, 조건 특정 또는 시간적 방식으로 RNA 기능을 조절합니다. 최근 연구에 따르면 인간 게놈에는 1500개가 넘는 RBP가 있으며 이러한 RBP는 RNA 수명주기 전반에 걸쳐 RNA 안정성과 기능을 조절하는 필수적인 역할을 하는 것으로 추정됩니다. RBP는 mRNA splicing, stability, localization 및 translation을 포함한 전사 후 유전자 조절에서 중요한 역할을 합니다. RBP의 돌연변이는 암, 신경퇴행성 질환, 근이영양증, 심장 질환, 대사 장애, 자가면역 질환 및 기타 여러 질환과 관련이 있습니다.
Enhanced Cross-Linking & ImmunoPrecipitation (eCLIP) sequencing은 transcriptome 전체에서 단백질-RNA 상호작용을 조사하는 강력한 도구입니다. 이 기술을 통해 수백 개의 RBP의 RNA 결합 부위를 결정하여 RNA 조절이 다양한 질병의 진행에 어떻게 기여하는지에 대해 알 수 있게 되었습니다. 그러나 아직 성공적으로 eCLIP이 완료되지 않은 RBP가 1,000개가 넘는 것으로 예측됩니다. eCLIP은 기존 방법보다 RBP 결합 부위를 식별하는 성공적인 방법이었지만 이러한 조건에서 성공적으로 작동할 수 있는 항체를 식별하는 것은 어려운 일이었습니다. 이에 eCLIP의 선두 그룹인 Eclipsebio사와 항체 선두 그룹인 Cell Signaling Technology (CST)가 파트너쉽을 맺어 eCLIP에 검증된 항체를 사용하여 성공적인 eCLIP 실험을 신속하게 진행할 수 있는 방법을 제시합니다.
Why eCLIP-Validated Antibodies Matter
A successful eCLIP experiment is heavily dependent on the quality of the antibody. Commercial IP-validated antibodies tend to fail validation due to eCLIP’s stringent washes. Eclipsebio & Cell Signaling Technology (CST) have partnered to expertly validate CST® antibodies.
How Eclipsebio Validates CST Antibodies
Eclipsebio’s IP-antibody validation method assesses both IP efficiency (western blot) and RNA yield (biotin blot) after stringent eCLIP washes. Eclipsebio’s validation standards are used both when selecting an antibody and during the actual eCLIP experiment.
Western and biotin blot antibody validation data presented for 2 proteins of interest in HEK293 cells using Eclipsebio’s IP-antibody validation method. Ab 1 results in a failed validation test. Biotin blot data for Ab 1 indicates RNA smear begins at incorrect molecular weight (M.W.). Bands for Ab 1 are not present at the expected M.W. of the western blot data. CST-validated antibody, Ab 2, results in a passed validation test. Biotin blot data for Ab 2 contains robust RNA smears at the expected M.W. Bands for Ab 2 are present at the expected M.W. in the western blot data.
The RBP-eCLIP Workflow
RBP-RNA interactions are UV cross-linked. RNA is fragmented, and an RBP of interest is immunoprecipitated with a CST antibody. PCR amplifi cation is then used to obtain sufficient material for high-throughput sequencing.
eCLIP-identified targets can be verified in any of three ways:
1. Compare the results to published CLIP or RNA-IP-Seq data to confirm high-confidence RBP binding locations are identified as areas of significant read enrichment in the IP library compared to the control library.
2. Execute an eCLIP experiment using antibodies against different RBPs within the same protein complex as the binding locations should be similar.
3. Perform an eCLIP experiment with antibodies against different epitopes within the same RBP to confirm similar binding locations.
Elevated p110 read densities on two adjacent, opposite stranded Alu elements with a prominent dip in coverage at the tip of a predicted hairpin. Antibodies 1 and 2 bind to different epitopes on p110 and their identifi ed peaks strongly overlap and show similar characteristics, indicating a strong likelihood of a true positive binding event. The hairpin region is denoted by a red mark on the sequence tracks and corresponds with the hairpin region boxed on the folded RNA.
Each antibody is tested in duplicate and must generate an acceptable Irreproducible Discovery Rate, as previously described for ChIP-Seq data analysis1.
IP fold enrichment over control (SMInput) of histone RNAs using RBP-eCLIP against SLBP is reproducible across biological replicates. Each point indicates eCLIP fold-enrichment over paired SMInput for the CDS (circle) or 3’UTR (square) of genes profiled in independent bio[1]logical replicate SLBP eCLIP experiments. Histone genes are indicated in pink, with open circles indicating significantly enriched regions (fold-enrichment ≥ 4-fold, p-value ≤ 10-5 in eCLIP vs SMInput). Both CDS (R2 = 0.50) and 3’ UTR (R2 = 0.73) show significant correlation (p < 10-300, all significance determined by standard conversion of r values to t-statistic), and show enrichment at most histones.
REFERENCES 1. Van Nostrand E.L., et al. (2016) Nat Methods. 13(6):508-14. 2. Li, Q., et al. (2011) Ann. Appl. Stat. 5(3),1752-1779.
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