Previous experiments by Spector's team and two other groups indicated that MENε and MENβ RNAs were the critical elements for paraspeckle formation. "What wasn't clear was how the paraspeckles actually form and the dynamics of how the non-coding MEN RNAs help organize and maintain its structure," says Spector.
To address this question, the team developed an innovative approachspearheaded by CSHL postdoctoral fellow Yuntao (Steve) Mao and graduate student Hongjae Sunwooto peer into living cells and capture the real-time dynamics of the interactions among the set of molecules known to be involved in paraspeckle formation. The scientists engineered cells in which each of these playersthe MENε/β genes, the newly formed MEN RNAs, and the various paraspeckle protein componentseach carried a different colored fluorescent tag. The cells were also genetically manipulated such that the MEN genes could be switched on by exposing the cells to a drug.
The resulting movies shot by the Spector team, showed that within five minutes of switching on the MENε/β gene, individual paraspeckle proteins arrived and assembled at the sites of MEN RNA transcription. As the RNA transcripts accumulated, the fully functional paraspeckles enlarged in tandem and eventually broke away to cluster around the transcription sites.
"Our experiments show that it is the act of MEN RNA transcription alone that triggers paraspeckle formation and sustains them," says Spector. In the absence of transcriptional activ
|Contact: Hema Bashyam|
Cold Spring Harbor Laboratory