Ribosomes are complex ribozymes that interpret genetic information by translating messenger RNA (mRNA) into proteins. Natural variation in ribosome composition has been documented in several organisms, and can stem from several different sources. A key question is whether specific control over ribosome heterogeneity represents a mechanism by which translation can be regulated. We used RiboMeth-seq to demonstrate that differential 2'-O-methylation of ribosomal RNA (rRNA) represents a considerable source of ribosome heterogeneity in human cells, and that modification levels at distinct sites can change dynamically in response to upstream signalling pathways. Ablation of one prominent methylation, induced by MYC oncogene expression, resulted in altered translation of select mRNAs and corresponding changes in cellular phenotypes. Thus, differential rRNA 2'-O-methylation can give rise to ribosomes with specialized function. This constitutes a broader mechanism where the specific regulation of rRNA modification patterns fine-tune translation.
Hepatitis B virus (HBV) is a major cause of liver diseases, including hepatocellular carcinoma (HCC), and more than 650,000 people die annually due to HBV-associated liver failure. Extensive studies of individual promoters have revealed that heterogeneous RNA 5′ ends contribute to the complexity of HBV transcriptome and proteome. Here, we provide a comprehensive map of HBV transcription start sites (TSSs) in human liver, HCC, and blood, as well as several experimental replication systems, at a single-nucleotide resolution. Using CAGE (cap analysis of gene expression) analysis of 16 HCC/nontumor liver pairs, we identify 17 robust TSSs, including a novel promoter for the X gene located in the middle of the gene body, which potentially produces a shorter X protein translated from the conserved second start codon, and two minor antisense transcripts that might represent viral noncoding RNAs. Interestingly, transcription profiles were similar in HCC and nontumor livers, although quantitative analysis revealed highly variable patterns of TSS usage among clinical samples, reflecting precise regulation of HBV transcription initiation at each promoter. Unlike the variety of TSSs found in liver and HCC, the vast majority of transcripts detected in HBV-positive blood samples are pregenomic RNA, most likely generated and released from liver. Our quantitative TSS mapping using the CAGE technology will allow better understanding of HBV transcriptional responses in further studies aimed at eradicating HBV in chronic carriers.IMPORTANCE Despite the availability of a safe and effective vaccine, HBV infection remains a global health problem, and current antiviral protocols are not able to eliminate the virus in chronic carriers. Previous studies of the regulation of HBV transcription have described four major promoters and two enhancers, but little is known about their activity in human livers and HCC. We deeply sequenced the HBV RNA 5′ ends in clinical human samples and experimental models by using a new, sensitive and quantitative method termed cap analysis of gene expression (CAGE). Our data provide the first comprehensive map of global TSS distribution over the entire HBV genome in the human liver, validating already known promoters and identifying novel locations. Better knowledge of HBV transcriptional activity in the clinical setting has critical implications in the evaluation of therapeutic approaches that target HBV replication.
Ribosomes are complex ribozymes that interpret genetic information by translating messenger RNA (mRNA) into proteins. Natural variation in ribosome composition has been documented in several organisms, and can stem from several different sources. A key question is whether specific control over ribosome heterogeneity represents a mechanism by which translation can be regulated. We used RiboMeth-seq to demonstrate that differential 2’-O-methylation of ribosomal RNA (rRNA) represents a considerable source of ribosome heterogeneity in human cells, and that modification levels at distinct sites can change dynamically in response to upstream signalling pathways. Ablation of one prominent methylation, induced by MYC oncogene expression, resulted in altered translation of select mRNAs and corresponding changes in cellular phenotypes. Thus, differential rRNA 2’-O-methylation can give rise to ribosomes with specialized function. This constitutes a broader mechanism where the specific regulation of rRNA modification patterns fine-tune translation.
Translational regulation impacts both pluripotency maintenance and cell differentiation. To what degree the ribosome itself exerts control over this process remains unanswered. Accumulating evidence has demonstrated heterogeneity in ribosome composition in various organisms. 2'-O-methylation of rRNA represents an important source of heterogeneity, where site-specific alteration of methylation levels can modulate translation. Here we explore changes in rRNA 2'-O-methylation during mouse brain development and during tri-lineage differentiation of human embryonic stem cells. We find distinct alterations between brain regions, as well as clear dynamics during cortex development and germ layer differentiation. We identify a methylation site which impacts neuronal differentiation. Modulation of its methylation levels affects ribosome association of the Fragile X Mental Retardation Protein and translation of WNT pathway-related mRNAs. Together, the data reveals ribosome heterogeneity through rRNA 2'-O-methylation during early development and differentiation and suggests a direct role for ribosomes in regulating translation during cell fate acquisition.
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