Animal characteristics and husbandry

The NMRs used in this study were part of the well characterised Calico colony. The progenitors of these animals were collected in Kenya. All animals are microchipped at 90 days of age, providing each individual with a unique nine-digit identifying number. NMRs were housed in multi-chambered plexiglass burrow systems in animal rooms maintained at 28–30 °C and 30–50% relative humidity, in attempts to simulate climatic conditions in their native equatorial habitat. The animals were fed ad libitum with fruit and vegetables (bananas, apples, oranges, butternut squash, red bell pepper, romaine lettuce, cucumber, green beans, corn, carrots and red garnet yams) and supplemented with a high protein and vitamin enriched cereal (Pronutro, South Africa).

Animals, ranging from neonates to 22 years old, as well as pregnant females were euthanised using isoflurane and killed by cardiac exsanguination. Four to eight animals of both sexes were used in each age cohort (Table 1). Tissues were harvested from the embryos and animals of different ages and immediately flash frozen in liquid nitrogen and stored at -80 °C until analysed. Brain and spleen samples were used in these studies. We chose spleen because, as a lymphoid organ, it consists of large numbers of white blood cells. Most of the transcripts extracted from spleen will arise from B cells, T cells and mononuclear phagocytes and may allow a window on “inflammaging”, thought to be a prominent ageing mechanism (Zuo et al. 2019). Brain was chosen as splicing patterns are especially complex and functionally important in this organ (Karlsson and Linnarsson 2017), and dysregulated splicing is a feature of many neurodegenerative and neurodevelopmental diseases in brain (Nik and Bowman 2019). All animal use and experiments were approved by the Buck Institute Institutional Animal Care and Use Committee (IACUC) protocol number A10173.

Table 1 Details of animals used in the study. Shown here are the numbers and age ranges of naked mole rats included in each age category in the current study Full size table

Candidate genes selected for analysis

An a priori list of splicing factor candidate genes were chosen based on associations with ageing or senescence in multiple human ageing cohorts and in senescent primary human cell lines (Harries et al. 2011; Holly et al. 2013; Latorre et al. 2017; Latorre et al. 2018b). Some of the splicing factors in this list have also been shown to associate with lifespan in both mice and humans (Lee et al. 2016) and cognitive dysfunction in human populations (Lee et al. 2019b), or to be involved in molecular responses to dietary restriction (Lee et al. 2019a). The list of genes included the negative regulatory splicing factors Hnrnpa0, Hnrnpa1, Hnrnpa2b1, Hnrnpd, Hnrnph3, Hnrnpk, Hnrnpm, Hnrnpul2, the positive regulatory splicing enhancers Pnisr, Srsf1, Srsf2, Srsf3, Srsf6, Tra2b and the core components of the spliceosome Sf1 and Sf3b1. A further panel of alternatively spliced candidate genes where specific isoforms have known links to brain function/dysfunction (Lye et al. 2019) and/or have been demonstrated to be dysregulated in senescent astrocytes (Lye et al. 2019) or linked with cognitive dysfunction in human populations (Lye et al. 2019), and/or neurodegenerative disease were chosen for analysis. These were Aph1a, App, Aqp4, Gfap, Klotho, Mapt, Psen1 and Psen2. An additional panel of transcripts known to be associated with senescence were also used: Atm, Cdkn1a, Cdkn2a, Cdkn2b and Tp53. Table 2 lists all genes/isoforms tested in this panel, rationale for their inclusion and references to supporting literature. Isoform structures are given in Supplementary figure S1. Six endogenous control genes were also selected on the basis of stability in ageing human populations (Harries et al. 2011): Hprt1, Idh3b, Polr2a, Ppia, Tbp and Ywhaz, however assays to Hprt1 and Ywhaz were subsequently excluded due to poor assay performance.

Table 2 Cognition and senescence-related transcripts and isoforms used for expression analysis. Shown here are the transcripts and isoforms selected for analysis and a brief description of their function. Details of the type of AS event for each isoform, and the exons involved are provided in the references indicated Full size table

RNA extraction

Snap-frozen tissues were first treated with RNAlater™-ICE Frozen Tissue Transition Solution (ThermoFisher, Waltham, MA, USA) according to the manufacturer’s instructions, to allow handling of tissue without RNA degradation occurring due to thawing of sample. Tissue sections were placed in 1 mL TRI Reagent® Solution (ThermoFisher, Waltham, MA, USA) supplemented with the addition of 10 mM MgCl 2 to aid recovery of microRNAs (Kim et al. 2012). Samples were completely homogenised in a bead mill (Retsch Technology GmbH, Haan, Germany) at a frequency of 30 cycles per second for 15 min. Phase separation was carried out using chloroform. Total RNA was precipitated from the aqueous phase by means of an overnight incubation at − 20 °C with isopropanol. A total of 1.2 μl Invitrogen™ GlycoBlue™ Coprecipitant (ThermoFisher, Waltham, MA, USA) was added prior to incubation to aid pellet recovery. RNA pellets were ethanol-washed twice and re-suspended in 1× TE buffer, pH 8.0. RNA quality and concentration were assessed by NanoDrop spectrophotometry (NanoDrop, Wilmington, DE, USA).

Reverse transcription

For RTPCR and Sanger sequencing as described below, a pool of 1000 ng of total RNA taken from 10 samples (one sample chosen at random from each NMR age group) was reverse transcribed using EvoScript Universal cDNA Master kit (Roche LifeScience, Burgess Hill, West Sussex, UK) in single 20 μl reactions, according to the manufacturer’s instructions, with the exception of a change to the extension phase of the reaction: a step of 30 min at 65 °C was used instead of 15 min at 65 °C. Resulting cDNA samples were diluted to a final volume of 75 μl with dH 2 O, and repeated as necessary to provide sufficient template for all RTPCR and sequencing reactions carried out. For assay validation by standard curve, reactions as described above were carried out in quadruplicate and pooled prior to a 6-step 1:2 dilution series. For qPCR, 1500 ng of total RNA was reverse transcribed using SuperScript® VILO™ cDNA Synthesis Kit (ThermoFisher, Waltham, MA, USA) in 20 μl reactions, according to the manufacturer’s instructions (Thermofisher n.d.a, b). Resulting cDNA samples were diluted to a final volume of 150 μl with dH 2 O to ensure sufficient volume for all subsequent qPCR reactions.

Assay design

Quantitative real-time reverse transcriptase PCR (qPCR) assays to mouse splicing factor assays were obtained from Thermo Fisher (ThermoFisher, Waltham, MA, USA). Assay Ids are available on request. Assays to NMR splicing factors and alternatively expressed isoforms were custom designed to determine expression levels of all candidate mRNAs. Given that the current NMR genome is not completely annotated, this required a four-step design process, as follows:

(a) “In silico” sequence alignment: Where genes were not being tested for alternate isoform expression, the genome sequences of genes in question were obtained from the UCSC Genome Browser (Kent et al. 2002) for the following species: NMR, Guinea pig, Rat, Mouse and Human (see Supplementary Table S1 for assemblies used). Intronic sequences were removed and resulting “in silico-spliced” sequences manually aligned across all 5 species to identify areas of highest homology. Where feasible, exon boundaries within the regions of high homology were then selected as target areas for design of qPCR assays. Where alternate isoforms exist and were required to be assessed, evidence from previous literature was sought to identify the isoforms in question, with particular focus on differential function of specific isoforms (the nature of each splicing change and the precise exons involved are shown in Supplementary Figure S1, and referenced in Table 2). Dependent upon the species in which previous work had been carried out, one or more sequences were obtained from the UCSC Genome Browser (Kent et al. 2002) along with the NMR sequence. Once again, “in silico splicing” and manual alignment was carried out as described in the previous paragraph, however in these cases the areas of alternative splicing were selected as targets for assay design. (b) Validation of exon junction sequences: Prior to design of qPCR assays, sequence verification was carried out by conventional RTPCR amplification of the predicted fragments to confirm identity. In all cases, primers were designed to the NMR sequence, with the aim of amplifying the target areas as defined above and confirming amplicon identity based on expected size. Where alternative spliced isoforms were to be amplified (and where isoform structure allowed) multiple primer sets were designed to amplify each isoform individually. In the cases of Aph1a, App and Mapt, this was not possible therefore one set of primers was designed to amplify all possible isoforms along with a set of nested primers to isolate individual isoforms using gel electrophoresis and band-stab PCR (Harries et al. 2004). Primer sequences are given in Supplementary Table S2. Template cDNA for the RTPCR was created from a pool of mRNA from all NMR age groups. This pool was reverse transcribed as described above and used in PCR reactions using either Microzone Megamix Royal (Clent Life Science, Stourbridge, UK) or Platinum™ II Hot-Start Green PCR Master Mix (ThermoFisher, Waltham, MA, USA). Reaction conditions were set according to manufacturer’s instructions except in some cases where annealing temperatures and cycle numbers used were specific to each amplicon. These are given in Supplementary Table S2. Amplicons were then checked using gel electrophoresis, and those which matched the expected size were taken forward for the next steps. Of the genes chosen, App and the p21b isoform of Cdkn1a proved refractory to RTPCR amplification and size-based verification. In the case of App, the predicted sequence was used for the assay design. For p21b, sequence homology between the known (human/mouse) variants and the NMR genome assembly was too low to design an assay with any degree of confidence, so this isoform was omitted at this stage. (c) Sanger sequencing: Once amplicons had been verified by size, each was then sequenced to absolutely confirm the in silico-predicted spliced mRNA sequence. Sanger sequencing was carried out on the ABI 3730 platform using standard protocols. PCR primers were designed to incorporate M13 forward and reverse tag sequences to enable use of a common sequencing primer for all reactions (see Supplementary Table S2). Sequence verification was successful in all cases, however the NMR Psen1 sequence showed no evidence of either the 3 base insertion (ins TAC ) or the 4 amino acid (VRSQ) splice variants previously reported in human studies (De Jonghe et al. 1999; Janssen et al. 2000), while the NMR Psen2 sequence did not contain the exon 5 skipped (PS2V) splice variant seen in human and animal studies (Sato et al. 1999; Moussavi Nik et al. 2015). Consequently, both genes were omitted from further analysis. (d) Assay design and verification: Following sequence confirmation, TaqMan® assays were designed to target regions as defined above. Forward primer, reverse primer and reporter sequences of all assays designed are given in Supplementary Table S3. To determine assay efficiency and linearity, standard curves were created from qPCR data generated using a serial dilution of NMR cDNA (see “Reverse transcription” section above). Assay efficiencies and r2 values are given in Supplementary Table S3.

Quantitative reverse transcriptase real-time PCR

A total of 1.0 μl cDNA (reverse transcribed as indicated above) was added to a 5 μl qRTPCR reaction including 2.5 μl TaqMan® Universal Master Mix II, no UNG (ThermoFisher, Waltham, MA, USA) and 0.25 μl custom TaqMan® probe and primer mix (corresponding to 900 nM each primer and 250 nM probe). Reactions were run in triplicate on 384-well plates using the QuantStudio 12 K Flex Real-Time PCR System (ThermoFisher, Waltham, MA, USA). Amplification conditions were a single cycle of 95 °C for 10 min followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min. We first compared levels of splicing factor transcripts in spleen and brain tissue from young NMR (12 months) and young mice (3–4 months), followed by assessment of splicing factor expression and alternative splicing patterns of key senescence or brain-related genes in an extended series of NMR brain tissues from foetal stages to extreme old age (21 to 22 years).

Data preparation