Myg1 exonuclease couples the nuclear and mitochondrial translational programs through RNA processing

Abstract

Semi-autonomous functioning of mitochondria in eukaryotic cell necessitates coordination with nucleus. Several RNA species fine-tune mitochondrial processes by synchronizing with the nuclear program, however the involved components remain enigmatic. In this study, we identify a widely conserved dually localized protein Myg1, and establish its role as a 3'-5' RNA exonuclease. We employ mouse melanoma cells, and knockout of the Myg1 ortholog in Saccharomyces cerevisiae with complementation using human Myg1 to decipher the conserved role of Myg1 in selective RNA processing. Localization of Myg1 to nucleolus and mitochondrial matrix was studied through imaging and confirmed by sub-cellular fractionation studies. We developed Silexoseqencing, a methodology to map the RNAse trail at single-nucleotide resolution, and identified in situ cleavage by Myg1 on specific transcripts in the two organelles. In nucleolus, Myg1 processes pre-ribosomal RNA involved in ribosome assembly and alters cytoplasmic translation. In mitochondrial matrix, Myg1 processes 3'-termini of the mito-ribosomal and messenger RNAs and controls translation of mitochondrial proteins. We provide a molecular link to the possible involvement of Myg1 in chronic depigmenting disorder vitiligo. Our study identifies a key component involved in regulating spatially segregated organellar RNA processing and establishes the evolutionarily conserved ribonuclease as a coordinator of nucleo-mitochondrial crosstalk.

Figure 1.

Myg1 is a dual localized protein with a 3′ to 5′ RNA exonuclease activity. (A) Bubble plot between the enrichment score and negative log₁₀ of P value of enrichment by Gene Set Enrichment Analysis of dual localization proteins identified from The Human protein Atlas database. Size of the bubble indicates the number of genes. The Gene Ontology (GO) terms are colour-coded based on the overall category of the process involved. (B) Three dimensional structural model of Myg1 based on RecJ exonuclease (N-terminal) and the C-terminal region was modelled based on hybrid protocol using other templates with known structure. (C) Putative active site residues DHH (106–108) and conserved C-terminal Histidine 344 predicted to be involved in substrate selectivity are shown in the zoomed in image. In vitro exonuclease activity of wild type recombinant human Myg1 on (D) 35-mer RNA, (E) 35-mer single stranded DNA and (F) 35-mer double stranded DNA. All the substrates were 5′end-labeled with [γ-³²P] ATP and the activity was monitored at 0, 30, 60, 90, 120 min post addition of the enzyme. Lane C1 is a control reaction without the enzyme and lane C2 is a control reaction with heat-inactivated enzyme. (G) Kinetics of the exonuclease assays with wild type and mutant Myg1_H344Q on 35-mer RNA and 35-mer single stranded DNA. Data represents the kinetic plot with mean values ± S.E.M. across two replicates.

Figure 2.

Myg1 is a highly conserved protein involved in mitochondrial functions. (A) Phylogenetic analysis of Myg1 homologs across various organisms with a minimum query cover of 45% and presence of DHH motif is depicted as a circular dendrogram. (B) Spot assays for the wild type and myg1Δ strains on fermentative medium containing yeast extract, peptone and dextrose (YPD) medium and respiratory growth medium containing yeast extract, peptone and glycerol (YPG) medium. Spot dilutions starting from 1 O.D._600 nm cells were ten-fold diluted across the plate. (C) The bar graph shows the mean and individual data points of oxygen flux in pmol/min/OD_600 nm cells as a representation of respiratory capacity measured by Oroboros O2k instrument in Wild type and myg1Δ Saccharomyces cerevisiae (BY4742) cells. The experiment was performed with 3 biological replicates. (Student's t test, P < 0.0004). (D) The levels of ATP was measured using luminescence based method in Wild type and myg1Δ strains across three replicates. The bar graph represents mean and individual data points in the ATP represented as a percentage of the wild type cells (Student's t test, P < 0.001). (E) Spot assay of wild type and myg1Δ strains upon complementation with human Myg1 (hMYG1) or the catalytically inactive DHH mutant Myg1 (hMYG1_DHH-ALL). (F) Bar graph depicts the oxygen consumption rate (OCR) as pmoles of oxygen consumed/min/ O.D._600 nm of S. cereviasiae culture across two independent biological replicates (mean ± SEM) in wild type yeast, myg1Δ strain. The same is depicted for complementations in the myg1Δ background with wild type human MYG1 targeting to both mitochondria and nucleus (hMYG1), nuclear targetting human Myg1 (hMYG1_{No Mito}), mitochondrial targeting hMYG1 (hMYG1_{No Nuc}) or the catalytically inactive mutant hMYG1 (hMYG1_DHH-ALL) complemented myg1Δ yeast cells. Inset represents kinetics of oxygen consumption in various yeast strains, their raw traces and linear fit in one representative experiment is depicted.

Figure 3.

Myg1 localizes to nucleolus and mitochondrial matrix. (A) Myg1 co-localization with newly synthesized RNA stained with 5-ethynyl uridine (EU) (top right) in live B16 cells, later fixed and immunostained with Myg1 (bottom left) and the nuclear DNA is counter stained with DAPI (top left). RGB images of the maximal intensity projection of confocal sections of the nuclei are split into green, red and blue channels that reflect EU, Myg1 and DAPI staining respectively. Scale bar 10 μm. (B) Live B16 cells pulse chased with EdU (5-deoxyethynyl uridine), later fixed and immunostained with Myg1. RGB images of the maximal projection of the confocal sections of the nuclei are split into green, red and blue channels that reflect EdU, Myg1 and DAPI staining respectively. (C) Fractionation of B16 cells followed by detection of Myg1 by western blot analysis in nucleolus and mitochondria. Enrichment of fractions is ascertained by using antibodies to lamin (nuclear marker), GAPDH (cytosolic marker), nucleolin (nucleolar marker), Cox4 (mitochondrial marker) along with Myg1. (D) Mitochondria isolation and mitoplast preparation subjected to western blot analysis of VDAC1 present in mitochondrial outer membrane, SOD2 localizing to mitochondrial matrix along with Myg1. GAPDH was used to ascertain cytoplasmic contamination. (E) Relative intensity ratios of VDAC1 and Myg1 with respect to SOD2 in mitoplast is normalized to their relative ratio in mitochondria.

Figure 4.

Establishment of Silexoseq analysis and involvement of Myg1 in ribosomal RNA processing. (A) Schematic representation of the anticipated pattern of the read accumulation in Silexoseq (silencing of exonuclease and sequencing) analysis of a 3′-5′ exonuclease. The red lines indicate the pattern of transcripts upon silencing and in blue are the control pattern with an active 3′-5′ exonuclease. Upon normalizing the sequencing read counts at every base position, a peak would be observed in regions where exonuclease cleaves the transcript. Silexoseq analysis of (B) ITS-1, (C) ITS-2 regions of the ribosomal RNA upon Myg1 knockdown. (D) Northern blot analysis of control and Myg1 silenced B16 cells using probes that map to ITS-1 and ITS-2 regions. (E) The hybridization signal from 47S, 46S and 45S pre-rRNA is labeled as the ‘primary transcript plus’ (PTP) and 41S, 36S, 32S, 20S and 12S are represented as relative change with respect to control cells. Bars represent mean and individual data points across two replicates. (F) Fractionation of cycloheximide treated B16 cells on linear 10–50% sucrose gradient analysed for the rRNA followed by western blot analysis for Myg1 and RPL7. (G) Bar plot for the cell translation analysis performed by Click iT™ HPG Protein synthesis kit in B16 cells treated with Control and Myg1 siRNA. The scatter plot from three independent experiments (unpaired Student's t test, P < 0.0001).

Figure 5.

Myg1 cleaves NeMito transcripts. (A) Pathway enrichment analysis performed on the set of upregulated genes (fold change > 2) in Myg1 siRNA treated B16 cells. Negative logarithm of enrichment P value and the fold enrichment score; outputs from DAVID gene set enrichment analysis are plotted for processes with P value < 10⁻⁷. (B) Heatmap for the list of nuclear encoded mitochondrial genes (NeMito) that are upregulated in the enrichment analysis. Colour code outside the Heatmap represents specific process within the mitochondria. (C) Real time PCR analysis of NeMito Genes in B16 cells upon Myg1 silencing. The experiment was performed in 2 independent biological replicates and the data represented is the mean and individual data point. (D, E) Silexoseq analysis of abundantly expressed NeMito RNAs Ndufb7 and Ndufb11.

Figure 6.

Myg1 mediates selective RNA turnover in mitochondria and governs OXPHOS. (A) Silexoseq analysis of isolated mitochondria from control and Myg1 silenced cells was carried out. Representation of the heavy strand of mitochondria; coordinates in the x-axis represent the genomic coordinates (in 5′ to 3′ direction) in continuum and each of the genes are highlighted in different colors at the backdrop. (B) RNA decay analysis performed by EU labeling in B16 cells treated with control and Myg1 siRNA. (C) Western blot analysis of ND5, CO1, ATP6, CYTB along with Myg1 from the whole cell lysates of B16 cells normalized to tubulin levels in control and Myg1 silenced cells. (D) Flow cytometric analysis of B16 mouse melanoma cells treated with Control and Myg1 siRNA and stained with TMRE to check the mitochondrial potential and MitoTracker green to check the mitochondrial mass. Experiments were performed with three independent biological replicates and represented as mean and individual data points (students t test for mitotracker green staining means not significantly different, and for TMRE staining P value < 0.0001). (E) Oxygen consumption measured using Clark Electrode in control and Myg1 siRNA treated B16 mouse melanoma cells. The traces correspond to the level of oxygen at various time intervals after addition of cells to the Oroboros chamber normalized to the level of oxygen at the time of addition of cells (inset). Oxygen consumption rate (OCR) determined from the slope of the curve is depicted as pmoles of oxygen consumed per minute per 10⁷ of cells, calculated from two independent biological replicates with comparable Myg1 silencing represented as mean and individual data points (Student's t test, P < 0.01).

Figure 7.

Footprint of Myg1 is evident in the complex disorder vitiligo. (A) Expression signals for Myg1, across non-lesional and lesional epidermis in the vitiligo microarray is depicted as a paired scatter plot (Student's paired t test, P < 0.0001). (B) Pathway enrichment analysis performed on the set of upregulated genes in microarray (fold change > 2 and P value < 10⁻⁷) that compares lesional with the non-lesional epidermis of fifteen vitiligo subjects. Negative logarithm of enrichment P value and the fold enrichment score, outputs from DAVID gene set enrichment analysis are plotted for processes with P value < 10⁻⁷. (C) Heatmap of common genes upregulated between Myg1 silenced B16 cells and vitiligo microarray. Asterix are colour coded to map genes to processes elaborated in D. (D) Heatmap of mitoribosomes, OXPHOS and ribosome related genes that are upregulated in lesional compared to non-lesional vitiligo skin. (E) Schematic depiction of the dual role of Myg1 in nucleus and mitochondria.