microRNAs (miRNAs) are small non-coding RNAs that regulate gene expression by destabilizing mRNA targets and reducing their translation. A miRNA called miR-10b is known to be upregulated in various cancers, most strongly in malignant gliomas. In most other cancers, miR-10b drives metastasis, but in gliomas, it’s critical to tumor cell survival. Thus, miR-10b inhibition is considered a highly promising therapeutic strategy for these tumors.
The trouble is, normal ways of identifying miR-10b targets haven’t worked. In previous research, published in EMBO Molecular Medicine, researchers at Brigham and Women’s Hospital observed that miR-10b regulation is unconventional. The miRNA “seed” (nucleotides 2–7 at the 5′ end of a microRNA), normally the primary determinant of miRNA binding and targeting, is essential but insufficient to miR-10b activity (not the main determinant of miR-10b activity).
Instead, the researchers found, that miR-10b seems to regulate mRNA splicing. To follow up, they conducted a transcriptome-wide analysis of the miR-10b interactome, not confined to the predictions of bioinformatic algorithms or mapping to mRNAs.
Their results, described in Molecular Cancer, suggest exciting new approaches to research not just in glioma but in cancer more generally. The authors are Rachid El Fatimy, PhD, instructor, Yanhong Zhang, PhD, postdoctoral fellow, Anna M. Krichevsky, PhD, associate professor of Neurology/Neurobiology, and colleagues in the Department of Neurology at the Brigham.
miR-10b Binds to U6 snRNA
Using high-throughput RNA sequencing, the researchers discovered that U6 small nuclear RNA (snRNA), a core component of the spliceosomal machinery, is the major miR-10b binding partner in glioma cells. U6 snRNA is located predominantly, if not exclusively, in the nucleus of these cells.
miR-10b co-localized with U6 snRNA more commonly in glioma cells than in other cells. Furthermore, human glioblastoma tumors exhibited remarkable—in some cells almost perfect—co-localization between miR-10b and U6.
Effects of miR-10b
Overexpression of miR-10b reduced U6 levels as much as two U6 antisense oligonucleotides did. Conversely, an miR-10b inhibitor upregulated the levels. These effects were specific to U6; other small RNAs were not affected by miR-10b modulators. Similarly, miR-10b destabilized U6 and an miR-10b inhibitor promoted stabilization.
During splicing, U6 snRNA undergoes a series of conformational changes required for accurate interactions with other components of the spliceosome. Further experiments showed miR-10b binding interfered with U6 conformation. Overall, the study demonstrated that miRNA functions beyond mRNA targeting and may directly control major cellular machineries such as the spliceosome.
These effects of miR‑10b on U6 resulted in alternative splicing, including splicing of CDC42, one gene that is critical to glioma growth.
A Foundation for Advances in Research
This study identifies snRNA as a new class of miRNA targets. This first-ever expansion of miRNA-regulated networks beyond mRNA targets and long non-coding RNA targets suggests completely new directions for research into miRNA-based cancer therapies.