The level of expression of long noncoding RNAs in mammalian Genome and molecular functions

da | Giu 14, 2022 | Biologia Molecolare

Fig 1 – Schematic representation of the IncRNAs analysis in mammalian genome

 

Abstract

What is the level of expression of long non-coding RNAs (lncRNAs) in mammalian genomes and what are their functions? To answer these questions Johnsson et al. [1] in a recent paper using allele-specific single-cell RNA sequencing (scRNA-seq) described the transcriptional regulation of lncRNAs compared to coding genes.  They demonstrated that, differently from mRNAs, lncRNA expression is more heterogeneous due to low bursting frequency.   Interestingly, they found that lncRNAs expression regulates the bursting efficiency of nearby coding genes.  

Discussion

In March 2022, a group of researchers from Karolinska Institute in Stockholm published a computational research work on lncRNAs, their function in cell cycle progression and their association with apoptosis [1]. The lncRNAs are non-coding RNAs longer than 200 base pairs (bp). They are about as numerous as coding genes but are usually less expressed. They appear to lack catalytic activity and act as regulators of gene expression [2]. The function of few lncRNAs is known, which is why the authors of this study are looking for new innovative applications, in the field in which they could be involved.

First, the researchers performed single-cell transcriptomes [3] at allele specific to investigate the presence of lncRNAs in mouse fibroblasts. Then, the authors defined the level of expression of lncRNAs and messenger RNAs (mRNAs) across cells. The data they obtained showed that lncRNAs are less expressed than mRNAs and the low expression was mostly governed by a lower transcriptional burst frequency. More than 30% of lncRNAs showed a long gap (more than 24 h) between two consecutive transcriptional bursts on each individual allele.

The authors also proved that the interaction between lncRNAs and mRNAs can have an effect on transcriptional burst size and frequency, and they carried out investigations on the genomic organization identifying loci with divergent mRNA-mRNA pairs, mRNA-lncRNA pairs and unidirectional mRNA-transcribed promoters and they observed an increased in expression of divergently transcribed promoters for mRNA-mRNA and mRNA-lncRNA promoters compared to unidirectional transcribing promoters. By these results the author concluded that burst frequency is increased for divergent mRNA-mRNA and lncRNA-mRNA transcribing promoters with no consistent increase in burst size.

To investigate the function of lncRNAs, the authors performed an analysis on single-cell transcriptomes from asynchronously grown mouse fibroblasts to determine their expression during the cell cycle. Some lncRNAs showed a cell-cycle specific expression, making it possible to define a link between lncRNA expression in each cellular state and the cellular phenotype. The authors selected at least 2 highly ranked candidates of lncRNAs from each cell cycle phase excluded lncRNAs that overlapped with multiple other genes and proceeded with seven lncRNAs, Next the cell cycle progression was synchronized by serum starvazion, thymidine block or nocodazole treatment and validated by flow cytometry and quantitative PCR all the seven lncRNAs had the predicted cell cycle expression pattern measured by RTqPCR. Next, they generated individual lentiviral transduced cell line with stable hairpin RNA (shRNA) which induced knockdown for 3 of the candidates (wincr1, Lockd and A730056A06Rik representing candidates from each cell cycle phase) and performed an in depth investigation.

The authors then decided to investigate the function of three lncRNAs: Lockd, Wincr1 and A730056A06Rik the authors demonstrate that the first lncRNA, Lockd promotes the expression of the cell cycle regulator Cdkn1b gene.  Lockd, shRNA or anti-sense oligo (ASO) decreased cell growth analysed by colony formation. A similar result was also obtained for Wincr1, as after siRNA depletion, they observed a decrease in colony formation proportional to the lncRNA depletion. The third lncRNA, A730056A06Rik, is an antisense transcript to Rgma gene, which is involved in cell survival. Again, the authors observed an effect on colony formation by ASO-mediated knockdown, they observed unexpected decrease in colony-forming cells on ASO-mediated 730056A05Rik knock-down although they stated that the effects observed needed further evaluation.

lncRNAs also showed an effect on apoptosis. The authors analysed cells expressing genes involved   in growth arrest upon DNA damage by treatment with mitomycin C (MMC). They selected lncRNAs expressed in these cells. They first observed that few candidate lncRNAs were induced by MMC treatment. They then demonstrated that these lncRNAs knockdown sensitize to apoptosis. 

Conclusions

In conclusion, despite accumulating evidence that the majority of long non-coding RNAs in mammals are likely to be functional, only a relatively small proportion has been demonstrated to be biologically relevant. Although plant lncRNAs involved in these functions have not been reported yet, reason to believe that many of the unknown functions of lncRNAs can be realised through these modes. These mechanisms of lncRNA action in plants can provide directions for future research, and more functions of plant lncRNAs can be determined.

References

  1. Johnsson, P., Ziegenhain, C., Hartmanis, L., Hendriks, G. J., Hagemann-Jensen, M., Reinius, B., & Sandberg, R. (2022). Transcriptional kinetics and molecular functions of long noncoding RNAs. Nature Genetics, 54(3), 306–317. https://doi.org/10.1038/s41588-022-01014-1
  2. Perkel JM (June 2013). . BioTechniques (paper). 54 (6): 301, 303–4. “We’re calling long noncoding RNAs a class, when actually the only definition is that they are longer than 200 bp,” says Ana Marques, a Research Fellow at the University of Oxford who uses evolutionary approaches to understand lncRNA function
  3. Picelli, S. et al. Smart-seq2 for sensitive full-length transcriptome profiling in single cells.Nat. Methods 10, 1096–1098 (2013)

Makuissu Kountchou Arlette

Master Industrial Biotechnology student