Importance of the RNA secondary structure for the relative accumulation of clustered viral microRNAs.
Micro (mi)RNAs are small non-coding RNAs with key regulatory functions. Recent advances in the field allowed researchers to identify their targets. However, much less is known regarding the regulation of miRNAs themselves. The accumulation of these tiny regulators can be modulated at various levels during their biogenesis from the transcription of the primary transcript (pri-miRNA) to the stability of the mature miRNA. Here, we studied the importance of the pri-miRNA secondary structure for the regulation of mature miRNA accumulation. To this end, we used the Kaposi's sarcoma herpesvirus, which encodes a cluster of 12 pre-miRNAs. Using small RNA profiling and quantitative northern blot analysis, we measured the absolute amount of each mature miRNAs in different cellular context. We found that the difference in expression between the least and most expressed viral miRNAs could be as high as 60-fold. Using high-throughput selective 2'-hydroxyl acylation analyzed by primer extension, we then determined the secondary structure of the long primary transcript. We found that highly expressed miRNAs derived from optimally structured regions within the pri-miRNA. Finally, we confirmed the importance of the local structure by swapping stem-loops or by targeted mutagenesis of selected miRNAs, which resulted in a perturbed accumulation of the mature miRNA.
Architecture et Réactivité de l'ARN - UPR 9002, Institut de Biologie Moléculaire et Cellulaire du CNRS, Université de Strasbourg, 15 rue René Descartes, F-67084 Strasbourg Cedex, France.
Maud
M
Contrant
Architecture et Réactivité de l'ARN - UPR 9002, Institut de Biologie Moléculaire et Cellulaire du CNRS, Université de Strasbourg, 15 rue René Descartes, F-67084 Strasbourg Cedex, France a.fender@ibmc-cnrs.unistra.fr.
Aurélie
A
Fender
Architecture et Réactivité de l'ARN - UPR 9002, Institut de Biologie Moléculaire et Cellulaire du CNRS, Université de Strasbourg, 15 rue René Descartes, F-67084 Strasbourg Cedex, France.
Béatrice
B
Chane-Woon-Ming
Architecture et Réactivité de l'ARN - UPR 9002, Institut de Biologie Moléculaire et Cellulaire du CNRS, Université de Strasbourg, 15 rue René Descartes, F-67084 Strasbourg Cedex, France.
Ramy
R
Randrianjafy
Architecture et Réactivité de l'ARN - UPR 9002, Institut de Biologie Moléculaire et Cellulaire du CNRS, Université de Strasbourg, 15 rue René Descartes, F-67084 Strasbourg Cedex, France.
Valérie
V
Vivet-Boudou
Architecture et Réactivité de l'ARN - UPR 9002, Institut de Biologie Moléculaire et Cellulaire du CNRS, Université de Strasbourg, 15 rue René Descartes, F-67084 Strasbourg Cedex, France.
Delphine
D
Richer
Architecture et Réactivité de l'ARN - UPR 9002, Institut de Biologie Moléculaire et Cellulaire du CNRS, Université de Strasbourg, 15 rue René Descartes, F-67084 Strasbourg Cedex, France s.pfeffer@ibmc-cnrs.unistra.fr.
Sébastien
S
Pfeffer
D001402:B-Lymphocytes / Q000821:virology; D002460:Cell Line; D019288:Herpesvirus 8, Human / Q000235:genetics; D006801:Humans; D035683:MicroRNAs / Q000737:chemistry* / Q000378:metabolism; D016296:Mutagenesis; D009690:Nucleic Acid Conformation; D012367:RNA, Viral / Q000737:chemistry* / Q000378:metabolism
D035683:MicroRNAs; D012367:RNA, Viral