Albany 2015:Book of Abstracts
June 9-13 2015
©Adenine Press (2012)
Nonenzymatic Recombination of RNA: Detection of Novel Sequences by Next-generation Sequencing
According to the hypothesis of RNA world at the early stages of evolution RNA fulfilled functions of genetic information storage and of catalysis. It was suggested, that detached ensemble of different RNA molecules capable to self-replication could be a predecessor of cell. The necessary condition of appearance of such ensemble is the presence of diversity of RNA. Successive cleavage and ligation reactions of RNA are one of the foremost ways leading to their recombination. Spontaneous RNA cleavage occurs with SN2-mechanism and results in a formation of RNA fragments with 2',3'-cyclophosphate and 5'-OH. These groups may react with each other yielding novel RNA molecules.
In order to study nonenzymatic RNA recombination, occurring through successive cleavage and ligation steps, the model system consisting of two RNA fragments was used. The RNAs are 96 nts fragment of HIV-1 genomic RNA, comprising primer binding site (HIV-RNA) and 96 nts fragment of Influenza virus A/WSN33/H1N1 genomic RNA, encoding M2 protein (M2-RNA), prepared by in vitro transcription. Cleavage and ligation reactions, occurring in solution containing only magnesium ions (5 mM MgCl2, pH 8.0, 37 °C, 0, 3, 7, 30 days) may lead to the formation of a wide variety of recombinant molecules. Our tasks were to identify these recombinant molecules, find the most widespread ones and find out RNA structures promoting ligation reaction.
The formed pools of RNA incubated for 3, 7 and 30 days and control pool were used for preparing cDNA libraries according to the following scheme: adapter ligation, reverse transcription, PCR. Libraries were amplified on beads in emulsion PCR and then sequenced on SOLiD 5.500 platform.
In order to analyze the sequencing data concept of a ligation point was suggested. A group of the recombinant products (novel sequences) corresponds to each ligation point. Totally 102 749 novel sequences, corresponding to 2099 ligation points were identified in three libraries (3, 7 and 30 days of RNA incubation). Frequency of occurrence of ligation points is not accidental and differs dramatically from 1 to several thousands of sequences per library. Frequently found ligation points correlate with fragile sites of HIV and M2 RNAs, however, this correlation is not strict.
A variety of novel RNA sequences decreases from about 50 thousands in 3 days library to 5 663 in 30 days library. Each group of products from 3 days library includes several RNAs differing significantly in their structure and length (30-75 nts). Each group from 30 day library includes one or several similar RNAs (20-32 nts). Totally in 7-30 days successive cleavage/ligation leads to generation of short 20-32 nts hairpin-like RNAs with apical loop 4-6 nts. The most abundant ligation points remain the same in all three libraries.
Analysis of the secondary structures of novel RNA products shows that ligation reaction occurs preferably i) within apical loops 4-6 nts, rarely 8 nts in length; ii) within internal loops and bulges 3-6 nts in length and iii) occasionally within double-stranded RNA regions at a distance of one or two nucleotides from a single-stranded region.
RNA products corresponding to one ligation point in 3 day library contain variable and constant (core) parts in their structure, and the core structure remains the same in all libraries. Thus we can conclude that the structure which provide for efficient ligation of RNA fragments is the same for each ligation point.
This work received financial support from Russian Science Foundation grant No. 14-44-00068.
Yaroslav Y. Staroselets
Institute of Chemical Biology and Fundamental Medicine