organism: Homo sapiens
description: Some mitochondrial long noncoding RNAs (lncRNAs) are encoded by nuclear DNA, but the mechanisms that mediate their transport to mitochondria are poorly characterized. Using affinity RNA pulldown followed by mass spectrometry analysis, we found two RNA-binding proteins (RBPs), HuR and GRSF1, that associated with the nuclear DNA-encoded lncRNA RMRP and mobilized it to mitochondria. After RMRP was imported into mitochondria, GRSF1 bound RMRP and increased its abundance in the matrix. Loss of GRSF1 lowered the mitochondrial levels of RMRP, in turn suppressing oxygen consumption rates and modestly reducing mitochondrial DNA replication-priming. We tested if RMRP was implicated in the replication of mtDNA, specifically if the corresponding reduction in RMRP altered the primer-generating efficiency and accordingly the RNA-DNA transition sites. This was done by ligation-mediated PCR (LMPCR) to detect free 5-prime ends of the newly synthesized mtDNA (H-strand origin) in both WT and GRSF1KO HEK293. The amplified DNA sequence was examined by next-generation sequencing and identified discrete positions similar to those found previously as the major replication initiation sites, although other sites (at nt positions 220, 310, and 440) were not detected, possibly due to the short segments of amplified DNA used for the library (less than 300 bp). The normalized read counts were significantly lower at several of the major 5-prime end positions (at nt 186, 149, and 146; p = 0.0343, 0.007, and 0.0004, respectively) in GRSF1KO cells relative to WT cells, suggesting that primer generation by RMRP enzyme activity was lower and thus DNA replication efficiency was partly reduced in GRSF1KO cells.
attributes.tag: isolate
attributes.value: HEK293
attributes.tag: age
attributes.value: embryonic
attributes.tag: biomaterial_provider
attributes.value: Myriam Gorospe, LG, NIA IRP, NIH
attributes.tag: sex
attributes.value: female
attributes.tag: tissue
attributes.value: Kidney
attributes.tag: cell_line
attributes.value: HEK293 GRSF1 KO
attributes.tag: dev_stage
attributes.value: embryonic
attributes.tag: sample_type
attributes.value: cell culture
attributes.tag: treatment
attributes.value: GRSF1 KO
attributes.tag: BioSampleModel
attributes.value: Human