Genome-wide screening of NEAT1 regulators reveals cross-regulation between paraspeckles and mitochondria

Research teams led by Dr. Ling-Ling Chen at Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences provided a new insight on the correlation between nuclear body paraspeckles and mitochondria. This study, entitled as “Genome-wide screening of NEAT1 regulators reveals cross-regulation between paraspeckles and mitochondria” (DOI:10.1038/s41556-018-0204-2), was published on Nature Cell Biology in Sep. 24th.

 

Paraspeckle is a kind of nuclear body which is assembled from lncRNA Nuclear Enriched Abundant Transcript 1 (NEAT1) and Paraspeckles proteins (PSPs). In human genome, the NEAT1 locus produces two isoforms of NEAT1, the long isoform NEAT1_2 (23,000 nt) and the short isoform NEAT1_1 (3,700 nt). The transcription of NEAT1 and the NEAT1_2 isoform are essential for paraspeckle formation and maintenance. Previous studies showed that NEAT1/paraspeckles play multiple roles in gene regulation by sequestrating proteins (such as SFPQ upon immune stimuli) or mRNAs with inverted repeats (IR) in their 3′ UTRs. The sequestration of mRNAs in paraspeckles is dynamically regulated in response to cellular stresses and during circadian rhythm regulation. Meanwhile, proteasome inhibition could induce the formation of “sausage-like” paraspeckles. Despite these advances in understanding functional implications of NEAT1/paraspeckles, little is known about how the NEAT1 gene itself is regulated and how paraspeckles communicate with other cell compartments.

 

To fill the gap in understanding how NEAT1 itself is regulated, the researchers developed an endogenous NEAT1-promoter driven EGFP reporter HeLa cell line by TALEN-mediated insertion of the EGFP sequence to the downstream of the transcription start site (TSS) of NEAT1, termed as NEAT1^G-HeLa. The genome-wide RNAi screening of NEAT1^G-HeLa yielded 171 gene candidates that involved in regulating the expression of NEAT1. Surprisingly, nuclear encoded genes with reported roles in regulating mitochondrial functions (mitochondrial proteins) were enriched in these candidates. High-confidence of this endogenous promoter-based screening was confirmed by shRNA knockdown assay. Meanwhile, fluorescence in situ hybridization (FISH) followed by SIM observations showed that the number of paraspeckles in cells was largely correlated with the level of NEAT1 expression. Intriguingly, knockdown of mitochondrial proteins led to increased proportion of elongated paraspeckles, yet no detectable morphological changes was found upon disruption of other examined chromatin modulators or TFs, except ATF2. Moreover, fluorescence recovery after photobleaching (FRAP) revealed that elongated paraspeckles had reduced dynamic behavior of NONO and increased capability of mito-mRNA (mRNAs of nuclear-encoded mitochondrial proteins) sequestration. Further studies found that ATF2 was involved in regulating NEAT1 by functioning as transcription factor (TF) in response to mitochondrial defects mediated by both mitochondrial proteins knockdown and stimuli induced by mitochondrial stressors.

 

To further clarify the cross-regulation between paraspeckles and mitochondria, the researchers found that altered NEAT1 expression mediated by CRISPR/Cas9, antisense oligos (ASOs) and dCas9-VP64 led to impaired mitochondria homeostasis. Mitochondrial morphology, oxygen consumption rate and ATP synthesis ability were impaired under aberrant NEAT1 expression, which ultimately affected the cell proliferation rate. In addition, NEAT1/paraspeckles protected cells from apoptosis upon treatment of mitochondrial stressor sodium arsenate by sequestration of mito-mRNAs cycs, and NEAT1 KO cells exhibited enhanced nucleocytoplasmic export of paraspeckle-enriched mito-mRNAs. These data further illustrated the notion that hundreds of mito-mRNAs being enriched in paraspeckles are likely play roles in coordinating the crosstalk between these two sub-cellular compartments.

 

Nature Cell Biology also published the review article named “A critiques of A mitochondria -- paraspeckle crosstalk” (https://rdcu.be/7Fx7), written by Archa Fox from The University of Western Australia, highly appraising the study above.

 

This study uncovered a cross-regulation between paraspeckles and mitochondria, which represents one type of bi-directional signaling. This study was financially supported by the National Natural Science Foundation of China, Chinese Academy of Sciences, the Ministry of Science and Technology of China, and the Howard Hughes Medical Institute.