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Characterization of a new class of RNA products from the Pax-5 oncogene: circular RNAs

ABSTRACT: Circular RNAs (circRNAs) represent a relatively new class of RNA products, which are gaining interest due to their involvement in many biological processes and cancer disease. Due to their innate ability to bind and modulate co-interacting proteins and microRNAs (miRs), circRNAs represent an additional layer of complex intracellular signaling and regulatory networks supporting cell biology and cancer phenotypes. In this study, we identify and characterize various circRNA products, which derive from the Pax-5 oncogene (circPax-5) in B lymphocytes. We establish the expression profiles of multiple circPax-5 variants in a collection of previously reported Pax-5 expressing tissues in addition to cell models and clinical samples from B cell cancer lesions. For example, we observe that circPax-5 products are overexpressed (up to 13-fold) in chronic lymphocytic leukemia (CLL) where the majority of these circRNAs consists of a specific circPax-5 isoform containing the sequence of exons 2 to 5 (circPax-5_2-5) of the Pax-5 locus. Given the established role of Pax-5 gene products in cancer biology, we also we set out to assess the impact of circPax-5 in cancer processes using conditional expression of circPax-5_2-5 in B cell lines. Interestingly, we found that circPax-5_2-5 was associated with cancer cell growth rates, which were dependent on B cell differentiation and cancer subtypes. We also characterized mechanistic features of circPax-5 in B cell lines based on common molecular features of circRNAs (ex: miR sequestering and sponging). CircPax-5 complexes from B cells were thus isolated by circRNA precipitations and submitted to deep sequencing analysis to identify co-interacting miRs. In addition to miR sponging profiles, circPax-5 were also studied for potential translation of novel peptides through putative internal ribosome entry sites (IRES). Using a dual IRES-dependent reporter gene assay, we found that circPax-5_2-5 harbors IRES motifs capable of encoding uncharacterized proteins. Altogether, our study introduces a new category of Pax-5 gene products which provide an extension of Pax-5-dependant signaling and function in B lymphocytes. This is particularly important in cancer cells given our observations of elevated circPax-5 levels in specific B cell cancer lesions. Furthermore, the elucidation of new players from the Pax-5 oncogene could provide the mapping of unknown cancer pathways, which could be targeted for therapeutic and/or diagnostic strategies in B cell cancer lesions.

ORGANISM(S): Homo sapiens

PROVIDER: GSE188623 | GEO | 2025/04/01

REPOSITORIES: GEO

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Project description:In platelets, splicing and translation occur in the absence of a nucleus. However, the integrity and stability of mRNAs derived from megakaryocyte progenitor cells remain poorly quantified on a transcriptome-wide level. As circular RNAs (circRNAs) are resistant to degradation by exonucleases, their abundance relative to linear RNAs can be used as a surrogate marker for mRNA stability in the absence of transcription. Here we show that circRNAs are enriched in human platelets 17-188 fold relative to nucleated tissues, and 14-26 fold relative to samples digested with RNAseR to selectively remove linear RNA. We compare RNAseq read depths inside and outside circRNAs to provide in silico evidence of transcript circularity, show that exons within circRNAs are enriched ~13X in platelets relative to nucleated tissues, and identify 3162 genes significantly enriched for circRNAs including some where all RNAs appear to be derived from circular molecules. We also confirm that this is a feature of other anucleate cells through transcriptome sequencing of mature erythrocytes, demonstrate that circRNAs are not enriched in megakaryocytes, and that linear RNAs decay more rapidly than circRNAs in platelet preparations. Collectively, these results suggest that circulating platelets have lost on aveage over 90% of their progenitor mRNAs, and that translation in platelets occurrs against the backdrop of a highly degraded transcriptome. Finally, we find that transcripts classified as products of reverse transcriptase template switching are both enriched in platelets and resistant to decay, countering the recent suggestion that up to 50% of rearranged RNAs are artefacts. A single rRNA depleted total RNA sample was sequenced. This together with 25 publicly available rRNA depleted total RNA samples (including 3 from platelets) were analysed using PTESFinder v 1 (http://sourceforge.net/projects/ptesfinder-v1/) to identify back-splice junctions, characteristic of circRNA transcripts. The contribution of circRNA producing exons was analysed on a gene by gene basis as follows: All circRNA transcripts identified in any sample were first pooled to define exons which can contribute to circRNA generation using custom scripts (available on request). For each sample, expression estimates (RPKMI) across all circRNA producing exons were computed for each locus using the total size of exons (in bp) and the read counts mapping to them. Similarly, total size and exonic read counts for exons for which no circRNA were detected in any sample were used to compute expression estimates (RPKME) for non-circRNA producing exons for each locus. Abundance ratios (RPKMI/RPKME and RPKMI/RPKMI+RPKME) were calculated and compared between Platelets and human tissues using Wilcoxon signed-rank test. Please note that the '25sample_info_accn_no.txt' contains the accession numbers and tissue/cell type information for 25 samples analyzed together.

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Characterization of a new class of RNA products from the Pax-5 oncogene: circular RNAs

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