Genetic configuration UAS/GAL4 system for RNAi mediated depletion of cg3165 and expression of human TREX1 transgenes in D. melanogaster Females from GAL4 driver lines listed in 1st column were crossed to males shown in the upper row.

The resulting copy numbers of fly cg3165 and human TREX1 content of the progeny is described.

Schematic presentation of generated transgenic constructs and their confirmation after genomic integration and activation.

(A) Genomic region containing cg3165 gene shown in GEP UCSC browser snapshot with selected tracks indicating protein domain, regulatory regions, and RNA-seq coverage. cg3165 CDS shown as thick blue blocks; it was replaced by GAL4-lox-dsRed-lox sequence shown between dashed lines (functional elements are not in scale). Red arrows indicate PCR primers used to confirm cg3165 CDS removal. (B) PCR results confirm the removal of CDS fragment. (C) Transgenic constructs used for establishment of UAS-hTREX1 and UAS-hTREX1 V235G fs Drosophila stocks. Black arrows indicate RT-PCR primers. Primers used for quantitative RT-PCR are shown in green. (D) RT-PCR results confirm hTREX1 and hTREX1 V235G fs expression. Genotypes are shown below the gel images. Act42A gene was used as a reference. Upper panel represents ‘no reverse transcription’ (-RT) control. First lane is the 1 kb Plus DNA Ladder. Primers’ targets are indicated on the right. (E) Quantitative RT-PCR results demonstrate higher expression levels of both TREX1 transgenes when driven by ubiquitous Act5C-GAL4 driver, compared to cg3165-GAL4 driver and show elevated transcription levels of hTREX1 V235G fs compared to hTREX1. Expression levels were normalized to Act42A. Bar graphs represent means with SD; ** p< 0.01; **** p< 0.0001. This panel was created using BioRender.com.

cg3165 knock out affects D. melanogaster dorsal vessel functional parameters.

(A) Cross-section and M-mode OCM images of WT and cg3165 KO-GAL4 flies (7 days old males shown). Yellow scale bar is 50 um. (B) End diastolic area (EDA) measurements. (C) End systolic area (ESA)measurements. (D) Fractional shortening (FS) parameters. One week and 5 weeks old flies shown in panels B-D; males are shown as blue shade boxes, females shown as red shade boxes. 1 week old sample sizes: WT males n=30, WT females n=29; cg3165 KO-GAL4 WT males n=30, KO-GAL4 WT females n=26. 5 weeks old sample sizes: WT males n=34, WT females n=35; cg3165 KO-GAL4 males n=36, cg3165 KO-GAL4 females n=34. Statistical significance shown as black brackets. * - p< 0.05; ** - p< 0.01; *** - p< 0.001; **** - p< 0.0001. Panels B and D were created using BioRender.com.

EDA reduction, behavioral impairment, and reduced survival probability observed in CG3165 KO flies are restored by transgenic expression of human TREX1 variants.

(A) One-week-old males’ EDA is significantly larger in cg3165> yw, cg3165> TREX1, and cg3165> TREX1 V235G fs than in cg3165 KO. Five-week-old males carrying hTREX1 and hTREX1 V235G fs demonstrate reduction of EDA compared to cg3165> yw. (B) One-week-old females’ EDA is significantly larger in cg3165> yw, cg3165> TREX1, and cg3165> TREX1 V235G fs than in cg3165 KO. Five-week-old females carrying hTREX1 and hTREX1 V235G fs demonstrate reduction of EDA compared to cg3165> yw. WT-yw, KO-cg3165 KO-GAL4, KO/+ - cg3165> yw; hTREX1 - cg3165> hTREX1; hTREX1 V235G fs - cg3165> TREX1 V235G fs. 1 week old sample sizes: WT males n=30, KO males n=30, KO/+ males n=30, hTREX1 males n=26, hTREX1 V235G fs males n=25; WT females n=29, KO females n=26, KO/+ females n=30, hTREX1 females n=26, hTREX1 V235G fs females n=29. 5 weeks old sample sizes: WT males n=34, KO males n=36, KO/+ males n=35, hTREX1 males n=36, hTREX1 V235G fs males n=34; WT females n=35, KO females n=34, KO/+ females n=36, hTREX1 females n=31, hTREX1 V235G fs females n=34. (C) cg3165 knock-out males show impaired locomotor behavior compared to WT (yw); hemizygous cg3165 (cg3165> yw) males demonstrate significant improvement. hTREX1 introduction (cg3165> hTREX1) results in climbing ability improvement comparable to WT levels. Males carrying RVCL-S associated hTREX1 V235G fs (cg3165> TREX1 V235G fs) demonstrate partial climbing ability restoration. (D) cg3165 knock-out females show impaired climbing compared to WT (yw). Normal locomotor behavior is observed in cg3165> yw, cg3165> TREX1, and cg3165> TREX1 V235G fs animals. Bar graphs (A-B) show means with SD. (E) Kaplan-Meier survival curves of cg3165 KO, cg3165 >yw, cg3165 >hTREX1 and cg3165 > hTREX1 V235G fs males. Survival rates of cg3165 knock-out males are significantly reduced. Upon introduction of 1 copy of cg3165 (cg3165 >yw) and transgenic hTREX1 (cg3165 >hTREX1), or hTREX1 V235G fs (cg3165 > hTREX1 V235G fs) the survival probability is increased. (F) Kaplan-Meier survival curves of cg3165 KO, cg3165 >yw, cg3165 >hTREX1 and cg3165 > hTREX1 V235G fs females. Survival rates of cg3165 knock-out females are significantly reduced. Upon introduction of 1 copy of cg3165 (cg3165 >yw) and transgenic hTREX1 (cg3165 >hTREX1), or hTREX1 V235G fs (cg3165 > hTREX1 V235G fs) the survival probability is increased. hTREX1 introduction has the strongest impact on longevity; hTREX1 V235G fs effect is the weakest. Statistical significance shown as black brackets.: * - p< 0.05; ** - p< 0.01; *** - p< 0.001; **** - p< 0.0001. Panels A–D were created using BioRender.com.

Over-expression of RVCL linked hTREX1 V235G fs reduces survival probability, movement impairment, and decrease of dorsal vessel EDA.

In aged flies, over-expression of ‘healthy’ hTREX1 transgene negatively affects climbing ability and results in smaller EDA. (A) Kaplan-Meier survival curves of Act5C>yw (blue line), Act5C>hTREX1 (orange line) and Act5C >hTREX1 V235G fs (yellow line) males. (B) Kaplan-Meier survival curves of Act5C>yw (blue line), Act5C>hTREX1 (orange line) and Act5C >hTREX1 V235G fs (yellow line) females. C- Climbing assay results in Act5C>yw, Act5C>hTREX1, Act5C >hTREX1 V235G fs males. D- Climbing assay results in Act5C>yw, Act5C>hTREX1, Act5C >hTREX1 V235G fs females. Bar graphs (C, D) represent means with SEM. (D) End Diastolic Area (EDA) measurements of Act5C>yw, Act5C>hTREX1, Act5C >hTREX1 V235G fs males. (E) End Diastolic Area (EDA) measurements of Act5C>yw, Act5C>hTREX1, Act5C >hTREX1 V235G fs females. 1 week old groups sample sizes: Act5C>yw males n=29, Act5C>hTREX1 males n=29, Act5C >hTREX1 V235G fs males n=28; WT females n=30, hTREX1 females n=29, Act5C >hTREX1 V235G fs females n=26. 5 week old groups sample sizes: Act5C>yw males n=25, Act5C>hTREX1 males n=32, Act5C >hTREX1 V235G fs males n=30; Act5C>yw n=35, Act5C>hTREX1 females n=36, Act5C >hTREX1 V235G fs females n=34. Statistical significance shown as black brackets. * - p< 0.05; ** - p< 0.01; *** - p< 0.001; **** - p< 0.0001. Panels C–F were created using BioRender.com.