UcallM® Ribosome Profiling Library Kit

UcallM® Ribosome Profiling Library Kit is a high-resolution translatome library construction kit for ribosome footprint sequencing (Ribo-seq). By precisely capturing ribosome-mRNA complexes during active translation elongation, it obtains ribosome-protected fragments (RPFs) to analyze translation dynamics, open reading frames (ORFs), nascent peptide chains and other information. Through technical optimization, Epibiotech efficiently and specifically removes rRNA and improves detection sensitivity for target RNA. The proportion of rRNA in sequencing data averages 14%, with a minimum of 5%. This kit is applicable to multiple mammalian species for studies on translational regulation, novel ORF discovery, or exploring drug effects on translation.

RNA-seq library kit needs to be purchased separately or prepared by users

Experimental Workflow

Application Scenarios 1.Profile ribosome distribution and translational activity on transcripts 2.Predict translation initiation sites and ORF positions 3.Determine protein translation efficiency 4.Explore translational regulation and gene expression 5.Identify novel proteins / novel peptides

Research Directions 1.Cancer research: aberrant translational regulation, tumor-specific antigen recognition, therapeutic resistance research 2.Disease mechanism research: translational efficiency profiling, non-canonical translation region discovery 3.Drug development & evaluation: target discovery, off-target effect detection, drug mechanism research Measured Data

Figure 1-60. Read length distribution of reads mapped to the genome

Figure 1-61. Length percentage of 5’UTR, CDS and 3’UTR

Figure 1-62. Usage frequency of different codons

Figure 1-63. Distribution of P-site signals across 5’UTR, CDS and 3’UTR regions Customer Publications 1.Cancer Commun: NAT10-Mediated ac4C Modification Drives Hepatocellular Carcinoma Progression by Promoting HMGB2 Translation via eEF2 (DOI:10.1002/cac2.12595) This study aimed to explore the functional mechanism of ac4C modification and its related acetyltransferase NAT10 in hepatocellular carcinoma (HCC), and identify potential therapeutic targets. Ribo-seq was used to analyze the effect of NAT10 knockout on mRNA translation efficiency. Loss of NAT10 markedly reduced translation efficiency of ac4C-modified mRNA, especially decreased ribosome occupancy on HMGB2 mRNA while mRNA levels remained unchanged. This study revealed the mechanism by which NAT10 enhances HMGB2 translation via ac4C modification, and identified eEF2 as the “reader” of ac4C that directly binds modified sites to promote translation elongation.

Figure 1-64. Transcriptional and translational changes after NAT10 knockout (H); reduced translation efficiency of ac4C-modified mRNA upon NAT10 knockout (I)

Figure 1-65. Reduced ribosome occupancy on HMGB2 mRNA

2.Cancer Cell: Targeting m⁶A Reader IGF2BP2 Suppresses Acute Myeloid Leukemia by Regulating Glutamine Metabolism (DOI:10.1016/j.ccell.2022.10.004) This study investigated the oncogenic role and regulatory mechanism of m⁶A reader IGF2BP2 in acute myeloid leukemia (AML), focusing on its regulation of glutamine metabolism, and developed small-molecule inhibitors targeting IGF2BP2 as potential therapeutic strategies. Ribo-seq was applied to analyze the effect of IGF2BP2 knockout on global translation efficiency (TE) in AML cells. RPF and mRNA expression levels of IGF2BP2 target genes were detected to verify whether IGF2BP2 regulates these genes by promoting translation.

Figure 1-66. Cumulative frequency distribution shift of global translation efficiency upon IGF2BP2 KD, indicating decreased translational activity

Figure 1-67. RPF/mRNA ratios of IGF2BP2 target genes (MYC, GPT2, SLC1A5) markedly decreased, showing translation is directly regulated by IGF2BP2