New research from the K.V. Prasanth lab, in collaboration with scientists across the University of Illinois and other institutions, has revealed that gene positioning near nuclear speckles — key nuclear domains that promote gene expression — along with the mechanistic functions of the non-coding RNA MALAT1, may act as driving forces underlying hypoxia-responsive gene regulation, particularly in cancer contexts.
Their findings were published recently in Molecular Cell and Cell.
The research was spearheaded by Cell and Developmental Biology PhD student You Jin Song, who originally set out to investigate the molecular mechanism by which MALAT1 regulates hypoxia-responsive gene expression.
MALAT1 is highly abundant in cells, which is uncommon for a typical non-coding RNA, and is heavily associated with cancerous tumors. Previous studies from Prasanth, Professor of Cell and Developmental Biology and Horwitz Scholar, and others have shown that removing MALAT1 from tumors stunts their growth.
Solid tumors are often highly dense, which limits the formation of functional blood vessels and leads to insufficient oxygen delivery.
“As a result, distinct hypoxic niches develop within different regions of the tumor,” Prasanth said. “Tumor aggressiveness is closely linked to how effectively cancer cells adapt to and exploit hypoxic conditions. Cancer cells that can better activate and utilize hypoxia-response pathways tend to become more aggressive.”
Notably, hypoxia induces a strong upregulation of MALAT1, which in turn modulates the hypoxia-responsive splicing of numerous pre-mRNAs. Despite this, a fundamental question remains: what are the underlying mechanisms by which MALAT1 regulates the expression of hypoxia-responsive genes?
“While we knew the identity of some of those genes, we previously didn’t really know where those genes were located within the nucleus,” Song said. “We plotted the positions of the genes on a linear scale, and they seemed to be clustered in specific chromosome regions. We didn’t really understand why it had this nonrandom distribution.”
Further experiments revealed that the hypoxia-responsive genes tend to be positioned near nuclear speckles.
“It was only after we saw the 3D genome architecture that we understood, ‘Oh, these genes are close to nuclear speckles where MALAT1 and other speckle proteins are concentrated.’ When we made that connection, that was really the ‘wow’ moment,” she said. “And we already know that nuclear speckles are important for enhancing splicing and gene expression, so this could point towards a potential mechanism for MALAT1.”
Eventually, Prasanth and Song did find that molecular mechanism.
“Further downstream analyses demonstrated that MALAT1 modulates hypoxia-responsive splicing by facilitating the localization of a member of the splicing factor family of proteins — SR-family of splicing factors (SRSFs) — to nuclear speckles and by providing specificity to their interactions with pre-mRNA substrates,” Prasanth said. Nevertheless, the mechanistic basis of MALAT1-mediated control of this process remains unclear.
A productive collaboration with Rohit Pappu’s lab at Washington University led to the characterization of SRSF1-MALAT1 microphase condensates, which are required to maintain a critical concentration of SRSF1 near nuclear speckles for efficient pre-mRNA splicing.
“Although MALAT1 was previously implicated in splicing regulation, we found that it drives the formation of SRSF1 microphase condensates,” Song said. “This process enhances the recruitment of SRSF1 to transcriptionally engaged RNA polymerase II, ultimately promoting efficient co-transcriptional RNA splicing.
“Understanding its mechanism could lead to future studies that focus on the downstream targets or interactors of MALAT1 or new therapeutic strategies based on its mechanism.” Ultimately, understanding the hypoxia response of cancer cells is crucial to understanding cancer physiology, she said.
Several labs in MCB contributed to the project. The Auinash Kalsotra lab in the Department of Biochemistry provided key insights into splicing factor-RNA interactions. The Erik Nelson lab in the Department of Molecular & Integrative Physiology conducted experiments in murine models to define the role of MALAT1 in cancer progression. In the Department of Cell & Developmental Biology, Andrew Belmont’s lab helped to map the spatial positioning of genes near speckles, and Supriya Prasanth’s lab also contributed in multiple experiments.
Prasanth also acknowledged the generous support from various funding agencies, including the National Institute of General Medical Sciences/National Institutes of Health, the National Science Foundation Science and Technology Center for Quantitative Cell Biology, the Advanced Research Projects Agency for Health, and the Cancer Center at Illinois.
