Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2013 Jul;9(7):1919-25.
doi: 10.1039/c3mb70043g. Epub 2013 Apr 12.

Transcripts for combined synthetic microRNA and gene delivery

Neha Kashyap  1 , Bich Pham, Zhen Xie, Leonidas Bleris
Affiliations

Transcripts for combined synthetic microRNA and gene delivery

Neha Kashyap et al. Mol Biosyst. 2013 Jul.

Abstract

MicroRNAs (miRNAs) are a class of short noncoding RNAs which are endogenously expressed in many organisms and regulate gene expression by binding to messenger RNA (mRNA). MicroRNAs are either produced from their independent transcription units in intergenic regions or lie in intragenic regions. Intragenic miRNAs and their host mRNAs are produced from the same transcript by the microprocessor and the spliceosome protein complex respectively. The details and exact timing of the processing events have implications for downstream RNA interference (RNAi) efficiency and mRNA stability. Here we engineer and study in mammalian cells a range of synthetic intragenic miRNAs co-expressed with their host genes. Furthermore, we study transcripts which carry the target of the miRNA, thereby emulating a common regulation mechanism. We perform fluorescence microscopy and flow cytometry to characterize the engineered transcripts and investigate the properties of the underlying biological processes. Our results shed additional light on miRNA and pre-mRNA processing but importantly provide insight into engineering transcripts customized for combined delivery and use in synthetic gene circuits.

PubMed Disclaimer

Figures

Figure 1
Figure 1. Architectures and representative results
(a) A schematic representation of the backbone plasmid. The plasmid consists of a bidirectional promoter flanked by diverging minimal CMV promoters. We use rtTA-expressing HEK293 TET-On cells with saturating Dox concentration. (b) The miRNA is placed at the 3’UTR, 5’ UTR (with and without splice sites) and in an intron between two dsRed exons while keeping the microRNA target at the 3’UTR. Constructs are arranged according to the position of the miRNA (functional and mutant) in the dsRed mRNA. Eight additional plasmids were constructed without the miRNA target at the 3’UTR. (c) Representative microscopy and flow cytometry data of a control plasmid (without miRNA target).
Figure 2
Figure 2. Functional miRNAs and splice sites
(a) Density contour plots of the constructs with and without target. (b) Overlaid flow cytometry histogram of dsRed with (gray) and without (color) target for the three constructs. (c) Flow cytometry. Y axis is the ratio of the mean of dsRed over the mean of amCyan. Control is plasmid without miRNA. (d) Representative microscopy images of the 5’ UTR miRNA circuit, wild-type miRNA circuit and 3’UTR miRNA circuit (with and without miRNA target).
Figure 3
Figure 3. Mutant microRNAs and splice sites
(a) Density contour plots of the constructs with and without target. (b) Overlaid flow cytometry histogram of dsRed with (gray) and without (color) target for the three constructs. (c) Flow cytometry data. Y axis is the ratio of the mean of dsRed over the mean of amCyan. Control is plasmid without miRNA. (d) Representative microscopy images of the 5’ UTR miRNA circuit, wild-type miRNA circuit and 3’UTR miRNA circuit (with and without miRNA target).
Figure 4
Figure 4. Functional microRNAs without splice sites
(a) Density contour plots of the constructs (with and without miRNA target). (b) Representative microscopy images of the 5’ UTR miRNA circuit and 3’UTR miRNA circuit (with and without miRNA target). (c) Overlaid flow cytometry histogram of dsRed with (gray) and without (color) target for the two constructs. (d) Flow cytometry data. Y axis is the ratio of the mean of dsRed over the mean of amCyan. Control is plasmid without miRNA. (e) Titration of the concentration of the plasmid carrying the 5’UTR miRNA.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Bartel DP. MicroRNAs: Genomics, biogenesis, mechanism, and function. Cell. 2004;116(2):281–297. - PubMed
    1. Easow G, Teleman AA, Cohen SM. Isolation of microRNA targets by miRNP immunopurification. RNA. 2007;13(8):1198–1204. - PMC - PubMed
    1. Stark A, Brennecke J, Bushati N, Russell RB, Cohen SM. Animal microRNAs confer robustness to gene expression and have a significant impact on 3′ UTR evolution. Cell. 2005;123(6):1133–1146. - PubMed
    1. Bartel DP. MicroRNAs: Target recognition and regulatory functions. Cell. 2009;136(2):215–233. - PMC - PubMed
    1. Pillai RS, Bhattacharyya SN, Filipowicz W. Repression of protein synthesis by miRNAs: How many mechanisms? Trends Cell Biol. 2007;17(3):118–126. - PubMed

Publication types

LinkOut - more resources