Relationship Between RNA Poly(A) Tail And M~6A-Modification In Mouse Embryonic Stem Cells And 3T3 Cells

Epigenetic regulation caused by modification of DNA and protein has been comprehensive studied,but modification of RNA is still a less studied area.Up to now,there are over a hundred known RNA modifications.And in various fields,these post transcriptional modifications play a significant role in regulating RNA molecules.However,the location and function of these RNA modifications just has limited research.At the same time,due to the complex structure and function of RNA,it is challenging to study the biological effects of RNA modification.In eukaryotes,the cap-structure and poly(A)-tail has an irreplaceable role in transcriptional regulation.However,there are a host of modifications in messenger RNA(m RNA)that can be used to maintain the stability of m RNA.N6-methyladenosine(m~6A)has been found to be the most common type of post-transcriptional chemical modification in mammalian m RNAs and long non-coding RNAs(lnc RNAs).In both mammals and yeast,RNA m~6A preferentially occurs in both gene coding regions and 3’untranslated regions(3’-UTRs),implicating its fundamental roles in every aspect of post-transcriptional regulation,including splicing,stability,and translation.The poly(A)tails are non-templated homopolymeric sequences at the 3’-ends of most m RNAs and lnc RNAs.The presence of a poly(A)tail is required for m RNA nuclear transport,stability,and Translation.In the meanwhile,revealing the composition,length,regulation and function of poly(A)tail will bring new enlightenment to the study of many biological processes.With the rapid development of next-generation sequencing technology,the transcriptome has been studied extensively.However,with the exception of the detailed composition of poly(A)tails,complete poly(A)tail information is hard to obtain.To fill this gap,poly(A)inclusive RNA isoform sequencing(PAIso-seq)and the second generation PAIso-seq2 were successfully exploited.These techniques not only allow transcriptome-wide measurement of the poly(A)tail length,but also the interior of m RNA and 3’-end non-A residues along with the full-length c DNA.Due to the fast development of transcriptomics and the continuous improvement of sequencing technology,whether there is a correlation between different RNA modifications is a research topic worthy of deep exploration and has far-reaching significance.In this study,A series of bioinformatics analyses were completed by constructing a full-length RNA sequencing library containing poly(A)tail and incorporating m~6A specific methylated RNA immunoprecipitation(RIP)and PAIso-seq2 sequencing techniques.The length of poly(A)tail and non-A residues were analyzed,and the relationship between m~6A modification and poly(A)tails of m RNA and lnc RNA was finally analyzed We demonstrated that in both NIH 3T3 cells and mouse embryonic stem cells(ESCs),m~6A-modified m RNA transcripts were polyadenylated compared to that of the input together with longer poly(A)tails.In addition,we discovered that the proportion of m RNAs with non-A residues compared to that of the input is different.In ES cells,the proportion of m RNAs with non-A residues in m~6A RIP was much lower than input samples.On the contrary,the proportion of m RNAs with non-A residues was slightly lower in m~6A RIP than that in input samples in 3T3 cells with most of the comparisons not significant.These results indicate that the proportion of poly(A)tails containing non-A residues for m~6A-modified m RNAs is cell-type specific.At the same time,we also analyzed lnc RNAs,the proportion of transcripts with non-A residues was much less in m~6A-modified transcripts than in input samples,in both 3T3 and ES cells.Whether these observations also exist in other species,is an interesting topic to be explored in the future.In summary,the dynamics of the poly(A)tails of m~6A-modified m RNAs and lnc RNAs were analyzed as well as the interaction between m~6A modification and the poly(A)tails of m RNAs and lnc RNAs was demonstrated.Additionally,the data also contains full-length c DNA information in addition to m6A and poly(A)tail information,making it possible to dissect the interactions between m~6A and alternative splicing,alternative polyadenylation,poly(A)tail length,as well as non-A residues in poly(A)tails.Our study also provides an excellent example of the interaction between different post-transcriptional RNA modifications.It lays a good foundation for further exploring the functional coordination of the two most abundant RNA posttranscriptional modifications.It provides a significant reference value for further,to explore the functional coordination of the post-transcriptional RNA modifications.