A team headed by a biomedical scientist in the University of California, Riverside, has established a new RNA-sequencing system –“Panoramic RNA Display by Overcoming RNA Modification Aborted Sequencing,” or PANDORA-seq — which can help discover numerous modified small RNAs which were previously undetectable.
RNA plays an essential role in decoding the genetic information in DNA to maintain an organism’s life. It is generally called the intermediate molecule utilized to synthesize proteins from DNA. Cells are filled with RNA molecules in complex and diverse forms, two chief kinds being ribosomal RNA, or rRNA; and transfer RNA, or tRNA; which are included in the synthesis of proteins.
Small RNAs play essential roles in health and diseases, including diabetes, cancer, neurological disorders, and infertility. Cases of small RNAs are microRNA; piwi-interacting RNA, or piRNA; and tRNA-derived little RNA, or tsRNA. Small RNAs may get altered by compound groups and so acquire new purposes.
The maturation of high-throughput RNA sequencing technology — useful for analyzing the quantity and sequences of RNA at a biological sample — has uncovered a growing repertoire of small RNA populations that fine-tune gene expression and protect genomes.
“PANDORA-seq can be widely used to profile small RNA landscapes in various physiological and disease conditions to facilitate the discovery of key regulatory small RNAs involved in these conditions,” said Qi Chen, an assistant professor of biomedical sciences in the UCR School of Medicine, who led the study published today in Nature Cell Biology. “Modified small RNAs wear an ‘invisibility cloak’ that prevents them from being detected by traditional RNA-sequencing methods. How many such modified RNAs are there? What is the origin of their sequences? And what exactly is their biological function? These are questions PANDORA-seq may be able to answer.”
PANDORA-seq employs a stepwise enzymatic treatment to remove key RNA alterations, which takes the invisibility cloak used by the altered small RNAs.
“PANDORA-seq has opened Pandora’s box of small RNAs,” said Tong Zhou, a bioinformatician at the University of Nevada, Reno School of Medicine and also a co-corresponding author of this study. “We can now dance with these once invisible partners in the RNA ballroom.”
According to Chen, PANDORA-seq uncovers a surprising small-RNA landscape that is dominated by tsRNAs and rRNA-derived tiny RNAs, or rsRNAs, instead of microRNAs, that were previously believed to dominate many mammalian tissues and cells.
“With PANDORA-seq, we found unprecedented microRNA/tsRNA/rsRNA dynamics when somatic cells are reprogrammed to induced pluripotent stem cells, which are generated by adult cells and have properties similar to those of embryonic stem cells, making them capable of differentiating into all cell types of the body,” explained Sihem Cheloufi, an assistant professor of biochemistry in UCR plus a co-corresponding author of the paper. “Some tsRNAs and rsRNAs can impact protein synthesis and even affect embryonic stem cell lineage differentiation in embryonic stem cells.”
Chen clarified the current best-studied types of small RNAs in mammals are microRNAs, which are abundant in mammalian somatic cells and control the type and volume of proteins the cells make; and piRNAs, which are mainly expressed in the testis and regulate germ cell development.
“Currently, these small RNAs can be comprehensively profiled by high-throughput methods such as RNA sequencing,” he explained. “However, the widely used small RNA sequencing protocols have intrinsic limitations, which prevent certain modified small noncoding RNAs from being detected during RNA sequencing. PANDORA-seq overcomes these limitations.”
Junchao Shi, a doctoral student working in Chen’s laboratory and the research paper’s first author is enthusiastic about the use of PANDORA-seq.
“The new method could revolutionize the view of small RNA landscapes,” he explained. “Frankly, all previous studies using traditional RNA-sequencing may now need to be revisited.”
Cheloufi said the team now wishes to know how tsRNA/rsRNA are created, how they operate in stem cells, and the way in which they orchestrate cell fate decisions during development.
“Answers to these questions are timely to develop diagnostic tools, identify therapeutic targets, and advance regenerative medicine,” she explained.
While growing PANDORA-seq, Chen was reminded of the parable of the blind men and the elephant, which teaches truth is only revealed when various components come together.
“We sometimes forget the big picture, being focused on just a small part of it,” he said. “Perhaps the only way to arrive at total truth — the big picture — is to push against our boundary of knowledge and confirm the revealed truth with newly devised technology.”
“It is fascinating to observe down the lenses of a microscope in the lab the profound cell fate change during cellular reprogramming and differentiation,” said Reuben Franklin, a doctoral student in Cheloufi’s laboratory and a coauthor on the study. “But PANDORA-seq allows us to eavesdrop on the molecular players during these processes.”