Vertebrate mitochondrial genomes show extraordinary GC skew which results in the synthesis of RNAs prone to form G-quadruplexes (G4). Such RNAs, although mostly non-coding, are transcribed at high rate and swiftly degraded by an unknown mechanism. Comprehensive proteomic and transcriptomic approaches revealed that quasi-RRM protein GRSF1 together with the mitochondrial degradosome composed of RNA helicase hSuv3 and PNPase control the levels of G4 containing RNAs in mitochondria. When this machinery is inactivated non-coding mtRNAs are upregulated and novel, previously undescribed transcript, which we named tRNA-like, strongly accumulates. In vitro reconstitution experiments showed that GRSF is responsible for G4 RNA melting essential for degradosome-mediated decay. Overall design: There are 2 sets of data which correspond to two RNAseq studies (degradosome set and GRSF1 set). Each set includes experiments which were performed in triplicates. The aim of the first studies was to analyze the effect of inactivation of the mitochondrial degradosome components (hSuv3, PNPase) on mitochondrial RNA (mtRNA). In the second studies we examined influence of GRSF1 silencing of mtRNA. In both cases we analyzed long transcripts and small RNAs. Strand-specific library preparation procedures were applied. Ligation-based approach was used to analyze short RNAs (from ~20 to ~200 nucleotides) whereas longer RNAs (> ~100 nucleotides) were analyzed after their random fragmentation followed by reverse transcription primed with random oligomers (dUTP-based protocol). RNA was isolated from unfractionated cells using TRI-Reagent. Before preparation of long transcripts libraries total RNA was subjected to rRNA depletion. This step was omitted in preparation of libraries dedicated for analysis of short RNAs. Total RNA was used for preparation of small RNA libraries. Libraries from different studies (degradosome and GRSF1) were sequenced with the help of different Illumina sequencing platforms. Dysfunction of hSuv3 was achieved by inducible expression of its dominant-negative mutant form (hSuv3-G207V), which we shown to be equivalent to hSuv3 silencing (PMID: 19864255), whereas PNPase or GRSF1 were depleted by siRNA transient transfections. In the case of GRSF1 studies as controls we analyzed untreated cells and cells transfected with non-targeting siRNA. In the case of degradosome studies as controls we analyzed untreated cells and cells expressing siRNA-insensitive version of PNPase (rescue experiment).