Ribosomal RNA fragmentation into short RNAs (rRFs) is modulated in a sex- and population of origin-specific manner

Abstract

Background: The advent of next generation sequencing (NGS) has allowed the discovery of short and long noncoding RNAs (ncRNAs) in an unbiased manner using reverse genetics approaches, enabling the discovery of multiple categories of ncRNAs and characterization of the way their expression is regulated. We previously showed that the identities and abundances of microRNA isoforms (isomiRs) and transfer RNA-derived fragments (tRFs) are tightly regulated, and that they depend on a person's sex and population origin, as well as on tissue type, tissue state, and disease type. Here, we characterize the regulation and distribution of fragments derived from ribosomal RNAs (rRNAs). rRNAs form a group that includes four (5S, 5.8S, 18S, 28S) rRNAs encoded by the human nuclear genome and two (12S, 16S) by the mitochondrial genome. rRNAs constitute the most abundant RNA type in eukaryotic cells. Results: We analyzed rRNA-derived fragments (rRFs) across 434 transcriptomic datasets obtained from lymphoblastoid cell lines (LCLs) derived from healthy participants of the 1000 Genomes Project. The 434 datasets represent five human populations and both sexes. We examined each of the six rRNAs and their respective rRFs, and did so separately for each population and sex. Our analysis shows that all six rRNAs produce rRFs with unique identities, normalized abundances, and lengths. The rRFs arise from the 5′-end (5′-rRFs), the interior (i-rRFs), and the 3′-end (3′-rRFs) or straddle the 5′ or 3′ terminus of the parental rRNA (x-rRFs). Notably, a large number of rRFs are produced in a population-specific or sexspecific manner. Preliminary evidence suggests that rRF production is also tissue-dependent. Of note, we find that rRF production is not affected by the identity of the processing laboratory or the library preparation kit. Conclusions: Our findings suggest that rRFs are produced in a regimented manner by currently unknown processes that are influenced by both ubiquitous as well as population-specific and sex-specific factors. The properties of rRFs mirror the previously reported properties of isomiRs and tRFs and have implications for the study of homeostasis and disease.

Computational meta-analysis of ribosomal RNA fragments: potential targets and interaction mechanisms

“…We note that ∼99.5% of the CLASH rRFs from 45S pre-rRNA were contained within mature rRNA borders (Figure 2 ). This argues against random breakage of the pre-rRNA as the source of rRFs and is consistent with the results in numerous non-Ago samples ( 40 ). We tested whether the flanking regions of the rRNA genes or their antisense strands contributed to the rRF pool found in CLASH and found negligible amounts of reads aligned to the antisense rRNAs (0.28%) and the upstream flanking regions of rRNA genes (0.01%).…”

“…The most obvious difference was an overrepresentation of rRFs at the termini of mature rRNAs ( Supplementary Table S8 , Supplementary Figure S9 ), and this has also been reported in hundreds of human lymphoblastoid cell lines ( 40 ) and in other species ( 21 , 22 , 36 , 90 , 91 ). We observed considerable counts of rRFs at the 5′ end of the 28S rRNAs in all whole cell samples of HEK293 cells and HeLa.…”

“…Sequencing of human sRNAs containing 2′,3′-cyclic phosphate has detected rRFs produced from nuclear rRNAs under stress conditions ( 39 ), pointing to a potential cleavage by ANG or other endoribonucleases. A recent paper has reported rRFs in human lymphoblastoid cell lines samples ( 40 ) and linked variation of the rRF expression with ethnicity, sex and tissue.…”

On the expanding roles of tRNA fragments in modulating cell behavior

“…As with the tRFs, the rules remain the same with the exception of the prefix, which now is ‘iso-’ (instead of ‘tRF-’). More recently, we extended it to ribosomal RNA-derived fragments, which are also known as rRFs ( 51 ). The rules for generating license plates for rRFs remain the same with the exception of the prefix which is now ‘rRF-’.…”

NERD-seq: A novel approach of Nanopore direct RNA sequencing that expands representation of non-coding RNAs

“…Such rRNA fragments called rRFs, have been described as being capable of modulating rRNA transcription and function (68). As well, these fragments are important to cell survival and proliferation(69) in a sex, tissue, and population specific manner (70).…”