Mitochondrial polymorphism m.3017C>T of SHLP6 relates to heterothermy

Abstract

Heterothermic thermoregulation requires intricate regulation of metabolic rate and activation of pro-survival factors. Eliciting these responses and coordinating the necessary energy shifts likely involves retrograde signalling by mitochondrial-derived peptides (MDPs). Members of the group were suggested before to play a role in heterothermic physiology, a key component of hibernation and daily torpor. Here we studied the mitochondrial single-nucleotide polymorphism (SNP) m.3017C>T that resides in the evolutionarily conserved gene MT-SHLP6. The substitution occurring in several mammalian orders causes truncation of SHLP6 peptide size from twenty to nine amino acids. Public mass spectrometric (MS) data of human SHLP6 indicated a canonical size of 20 amino acids, but not the use of alternative translation initiation codons that would expand the peptide. The shorter isoform of SHLP6 was found in heterothermic rodents at higher frequency compared to homeothermic rodents (p < 0.001). In heterothermic mammals it was associated with lower minimal body temperature (T _b, p < 0.001). In the thirteen-lined ground squirrel, brown adipose tissue-a key organ required for hibernation, showed dynamic changes of the steady-state transcript level of mt-Shlp6. The level was significantly higher before hibernation and during interbout arousal and lower during torpor and after hibernation. Our finding argues to further explore the mode of action of SHLP6 size isoforms with respect to mammalian thermoregulation and possibly mitochondrial retrograde signalling.

Keywords: SHLP6; daily torpor; extended vertebrate mitochondrial genetic code; hibernation; micropeptide; mitochondrial-derived peptide (MDP); mitogenomics; rodents.

FIGURE 1

Phylogenetic tree depicting the distribution of SHLP6 peptide length across rodent species. Occurrence of heterothermy is highlighted in blue. 20 and 9mers of SHLP6 are depicted by a full bar outside of the tree or a half stroke, respectively. An additional short stroke highlights a species harbouring both length variants. In case no sequences were available at NCBI, the bar is left blank. Further research on little explored rodents is requested to increase the number of species with confidently classified as heterothermic (Ruf and Geiser, 2015). Statistic: chi-square test.

FIGURE 2

Length isoforms of SHLP6 in relation to physiological parameters of hibernation. Distribution of SHLP6 sizes across heterothermic mammals in relation to minimum T _b (A), minimum torpid MR (B) and ratio of minimal to basal MR with T _b and MR values taken from Ruf and Geiser (2015) (C). Analysis of significance: Wilcoxon rank-sum test, T _b : body temperature, MR: metabolic rate.

FIGURE 3

Sporadic detection of the non-enriched human SHLP6 using MS analysis (A) Hypothetical extension of the canonical human 20mer of SHLP6 predicted by Kienzle et al. (2023). This putative N-terminal extension is depicted by smaller font type. It was hypothesized to result from the use of the alternative translation initiation codons ATT and ATA depicted in violet. Positive or negative numbering refers to the canonical peptide or the hypothetical extension, respectively. The sequence of the cleaved 14mer peptide fragment, “MLDQDIPMVQPLLK”, is shaded. The two trypsin cleavage sites contained in the canonical sequence of human SHLP6, but not in the hypothetical N-terminal extension, are shown by the arrows. Origin of coding sequence: NCBI’s accession number J01415.2. (B) Details on the four human data sets that contained at least a single SHLP6-specific fragment. Notably, the same confident fragment was detected in all cases. (C) Example of a MS spectrum depicting the confident fragment “MLDQDIPMVQPLLK”. Blue and red signals represent “y” and “b” ions, respectively. Pre: precursor. The exemplary spectrogram was taken from the last of the data sets listed above.

FIGURE 4

Dynamic abundance alteration of SHLP6-encoding RNA in brown adipose tissue of the thirteen-lined ground squirrel during states of hibernation. (A) Deduction of SHLP6 ORF used as “query” sequence in the comparison of steady-state RNA levels. Deduction considered the extended coding repertoire of the mitogenome, here, the import of the cytoplasmic tRNA^Arg (AGG) into the mitochondrion (Rubio et al., 2008; Jeandard et al., 2019). As a result, the stop codon that divided the two peptides (in red font), gets converted and the putative SHLP6 peptide expanded. Amino acids are depicted by single-letter code and Rⁱ denotes the arginine residue resulting from import of cytoplasmic tRNA^Arg. (B) Temporal pattern of the steady-states of transcripts covering the extended SHLP6-encoding ORF. RNA abundance is expressed as transcripts per million. Error bars depict standard deviations. Wald test implemented by DESeq2 was used to assess significance of differential transcript abundance. Difference among a pair of time points is indicated by a different small or capital letter (small letter: 0.05 > p > 0.01, capital letter: p < 0.01). Accession number of the public RNA-seq data used for differential analysis of transcript abundance: PRJNA226612.

FIGURE 5

Features of SHLP6 (A) The sORF of SHLP6 occurs at two size variants exemplarily represented by human, rat and mouse. Polymorphism m.3,017C>T (red letter) introduces a stop codon in the mouse. In azure: hosting gene MT-RNR2. Arrow: sORF orientation. (B) Secondary structure predicted by RNAFold3 for the human MT-RNR2 transcript that hosts the sORF of SHLP6 (nucleotides 1,671 to 3,229 in NC_012920.1). m.3,017C>T depicted by the arrow does not affect folding of the hosting transcript. Dark blue: sORF of the 9mer variant of SHLP6 (30 nucleotides including stop codon); light blue: sORF of the 20mer variant of SHLP6 (63 nucleotides including stop codon). A similar secondary structure was predicted with UNAfold (data not shown). (C) Degree of hydrophilicity or hydrophobicity analysed for the amino acids of human and rat SHLP6 [hydropathicity plot in Kyte-Doolittle scale (Kyte and Doolittle, 1982)]. Note that the nine amino acids of mouse SHLP6 are shared by the two 20mer variants of the peptide (see sequence consensus). (D) Amino-acid sequences of the SHLP6 length isomers with information on charge (grey background) and putative PTM (SIM: SUMO-interacting motif, hexagon: methylation). Asterisk: identical amino-acid residue. We note that the contribution of the PTMs cannot be predicted in relation to daily or multiday torpor in silico.

FIGURE 6

Rodent SHLP6 structures with helices. Three-dimensional peptide structures as predicted by the artificial intelligence system AlphaFold2 are shown on the left with prediction confidence per region indicated by color. The secondary structure for each peptide is shown in the middle with alpha helices indicated in red. Amino acid sequences are shown on the right. All peptides with helices had a length of 20 amino acids. Positions five and 15 of the peptide are highlighted by an asterisk.

FIGURE 7

Rodent SHLP6 structures without helices. Three-dimensional peptide structures as predicted by AlphaFold2 are shown on the left with prediction confidence per region indicated by color. No secondary structures were predicted as shown by the horizontal, blue lines. Amino acid sequences are shown on the right. A total of three sequences had a length of 20 amino acids. Only the aligned parts of the peptides are shown. The asterisk highlights position five of the peptide.