Heintz C et al. (2016) Nature "Splicing factor 1 modulates dietary restriction and TORC1 pathway longevity in C. elegans."

Bruun GH, Andresen BS, Hoxhaj G, Zhang Y, Morantte I, Mair WB, Weir HJ, Lanjuin A, Heintz C, Doktor TK, Dutta S, Escoubas CC, Silva-Garcia CG, Manning BD

[Nature, 2016]

Ageing is driven by a loss of transcriptional and protein homeostasis and is the key risk factor for multiple chronic diseases. Interventions that attenuate or reverse systemic dysfunction associated with age therefore have the potential to reduce overall disease risk in the elderly. Precursor mRNA (pre-mRNA) splicing is a fundamental link between gene expression and the proteome, and deregulation of the splicing machinery is linked to several age-related chronic illnesses. However, the role of splicing homeostasis in healthy ageing remains unclear. Here we demonstrate that pre-mRNA splicing homeostasis is a biomarker and predictor of life expectancy in Caenorhabditis elegans. Using transcriptomics and in-depth splicing analysis in young and old animals fed ad libitum or subjected to dietary restriction, we find defects in global pre-mRNA splicing with age that are reduced by dietary restriction via splicing factor 1 (SFA-1; the C. elegans homologue of SF1, also known as branchpoint binding protein, BBP). We show that SFA-1 is specifically required for lifespan extension by dietary restriction and by modulation of the TORC1 pathway components AMPK, RAGA-1 and RSKS-1/S6 kinase. We also demonstrate that overexpression of SFA-1 is sufficient to extend lifespan. Together, these data demonstrate a role for RNA splicing homeostasis in dietary restriction longevity and suggest that modulation of specific spliceosome components may prolong healthy ageing.

Heintz C et al. (2017) Nature "Corrigendum: Splicing factor 1 modulates dietary restriction and TORC1 pathway longevity in C. elegans."

Andresen BS, Weir HJ, Manning BD, Mair WB, Lanjuin A, Morantte I, Heintz C, Doktor TK, Silva-Garcia CG, Bruun GH, Escoubas CC, Zhang Y, Dutta S, Hoxhaj G

[Nature, 2017]

This corrects the article DOI: 10.1038/nature20789.

Branicky R et al. (2017) Cell Res "Making a splash with splicing."

Hekimi S, Branicky R

[Cell Res, 2017]

In a recent Nature paper, Heintz et al. identify a splicing factor (SFA-1) that is crucial for the longevity conferred by dietary restriction and the TORC1 pathway modulation in C. elegans.

Dutta, S. et al. (2017) International Worm Meeting "Novel molecular mechanisms linking RNA splicing and longevity."

Mair, W., Heintz, C., Dutta, S.

[International Worm Meeting, 2017]

Aging is driven by a loss of cellular and organismal homeostasis and is the key risk factor for multiple chronic diseases. Interventions that attenuate or reverse systemic dysfunction seen with age therefore have potential to ameliorate the comorbidities associated with old age and prolong healthy years in the elderly. Although loss of protein and transcriptional homeostasis are well-established causes of aging, the decline of RNA homeostasis and splicing fidelity with age remains poorly understood. A recent study in the lab has demonstrated pre-mRNA slicing fidelity as a biomarker and predictor of life expectancy in C. elegans. Using both in vivo alternative splicing reporters and molecular genetics, we have shown that dietary restriction mediated extension of lifespan require specific components of the splicing machinery. However, these factors act downstream of different longevity pathways. REPO-1 was specifically required for lifespan extension in the electron transport chain mitochondrial mutants isp-1(qm150) and clk-1(qm30) but had no effect on longevity through the TORC-1 pathway. On the other hand, SFA-1 was shown to be required for longevity through the TORC1 pathway. This suggests that in addition to the broad dysfunction of splicing with age, alternate pathways that regulate longevity could be influenced by differential composition of the spliceosome. Lastly, overexpression of these splicing factors is sufficient to extend lifespan of wild type nematodes. Thus, understanding the role of these specific splicing factors in modulating alternate longevity paradigms would not only provide information about the mechanisms of these longevity pathways but will also give us new therapeutic windows to multiple age-related pathologies.

Howell, P. et al. (2019) International Worm Meeting "Molecular mechanisms linking pre-mRNA splicing to dietary restriction and TORC1 mediated lifespan extension."

Heintz, C., Howell, P., Lanjuin, A., Dutta, S., Mair, W.

[International Worm Meeting, 2019]

Aging is driven by the loss of cellular homeostasis and is the largest risk factor for multiple chronic diseases. Interventions including dietary restriction (DR), without malnutrition, and suppression of target of rapamycin 1 (TORC1), delay aging and protect against multiple age-related chronic diseases but the mechanisms are unknown. Most work into the mechanisms of DR have focused on transcriptional and proteomic maintenance while the decline in RNA homeostasis with age remains poorly understood. Our previous work has identified a new link between RNA homeostasis and aging. We have shown that specific components of the RNA splicing machinery, including branchpoint binding protein (BBP)/splicing factor 1 (SFA-1), are required for dietary restriction and TORC1 mediated lifespan extension in C. elegans. However, mechanisms linking RNA splicing to TORC1, DR and longevity are unknown. Here we examine the regulation of SFA-1 in TORC1 and DR mediated longevity. Phosphoproteomic data suggest that the mammalian ortholog of SFA-1, SF1 is differently phosphorylated with mTORC1 suppression in mammalian cell culture. In yeast, these two conserved serine phosphorylation sites are regulated by DSK-1, the mammalian ortholog of SR protein kinase 1. We will present ongoing work investigating the requirement of the worm ortholog of SR kinase 1, spk-1, in SFA-1 function and TORC1 mediated lifespan extension. In addition, we have utilized site specific CRISPR/Cas9 gene editing to directly mutate the serine sites in SFA-1 to block or mimic phosphorylation. This work will determine whether these phosphorylation sites are critical for SFA-1 activity and impact TORC1 mediated longevity in C. elegans. Furthermore, we will examine the impact of TORC1 suppression and DR on SFA-1 activity in mammalian systems. Together these data will determine a novel mechanism linking TORC1 signaling to longevity via regulation of splicing factor 1.

Silva-Garcia CG et al. (2019) G3 (Bethesda) "Single-Copy Knock-In Loci for Defined Gene Expression in Caenorhabditis elegans."

Dutta S, Heintz C, Mair WB, Silva-Garcia CG, Lanjuin A, Clark NM

[G3 (Bethesda), 2019]

We have generated single-copy knock-in loci for defined gene expression (SKI LODGE) system to insert any DNA by CRISPR/Cas9 at defined safe harbors in the <i>Caenorhabditis elegans</i> genome. Utilizing a single crRNA guide, which also acts as a Co-CRISPR enrichment marker, any DNA sequence can be introduced as a single copy, regulated by different tissue-specific promoters. The SKI LODGE system provides a fast, economical and effective approach for generating single-copy ectopic transgenes in <i>C. elegans</i>.

Wester LE et al. (2023) Cell Rep Methods "A single-copy knockin translating ribosome immunoprecipitation toolkit for tissue-specific profiling of actively translated mRNAs in C. elegans."

Mair WB, Lanjuin A, Denzel MS, Stine PG, Perez-Matos MC, Wester LE, Heintz C, Bruckisch EHW

[Cell Rep Methods, 2023]

Here, we introduce a single-copy knockin translating ribosome immunoprecipitation (SKI TRIP) toolkit, a collection of Caenorhabditis elegans strains engineered by CRISPR in which tissue-specific expression of FLAG-tagged ribosomal subunit protein RPL-22 is driven by cassettes present in single copy from defined sites in the genome. Through in-depth characterization of the effects of the FLAG tag in animals in which endogenous RPL-22 has been tagged, we show that it incorporates into actively translating ribosomes and efficiently and cleanly pulls down cell-type-specific transcripts. Importantly, the presence of the tag does not impact overall mRNA translation, create bias in transcript use, or cause changes to fitness of the animal. We propose SKI TRIP use for the study of tissue-specific differences in translation and for investigating processes that are acutely sensitive to changes in translation like development or aging.

Zhao Y et al. (2007) BMC Genomics "Poly-G/poly-C tracts in the genomes of Caenorhabditis."

Zhao Y, O'Neil NJ, Rose AM

[BMC Genomics, 2007]

ABSTRACT: BACKGROUND: In the genome of Caenorhabditis elegans, homopolymeric poly-G/poly-C tracts (G/C tracts) exist at high frequency and are maintained by the activity of the DOG-1 protein. The frequency and distribution of G/C tracts in the genomes of C. elegans and the related nematode, C. briggsae were analyzed to investigate possible biological roles for G/C tracts. RESULTS: In C. elegans, G/C tracts are distributed along every chromosome in a non-random pattern. Most G/C tracts are within introns or are close to genes. Analysis of SAGE data showed that G/C tracts correlate with the levels of regional gene expression in C. elegans. G/C tracts are over-represented and dispersed across all chromosomes in another Caenorhabditis species, C. briggase. However, the positions and distribution of G/C tracts in C. briggsae differ from those in C. elegans. Furthermore, the C. briggsae dog-1 ortholog CBG19723 can rescue the mutator phenotype of C. elegans dog-1 mutants. CONCLUSIONS: The abundance and genomic distribution of G/C tracts in C. elegans, the effect of G/C tracts on regional transcription levels, and the lack of positional conservation of G/C tracts in C. briggsae suggest a role for G/C tracts in chromatin structure but not in the transcriptional regulation of specific genes.

Bhagwati P Gupta et al. (2002) West Coast Worm Meeting "Genetic analysis of the vulval mutants in C. briggsae"

Shahla Gharib, Bhagwati P Gupta, Paul W Sternberg, Jennifer X Li

[West Coast Worm Meeting, 2002]

To understand the evolution of developmental mechanisms, we are doing a comparative analysis of vulval patterning in C. elegans and C. briggsae. C. briggsae is closely related to C. elegans and has identical looking vulval morphology. However, recent studies have indicated subtle differences in the underlying mechanisms of development. The recent completion of C. briggsae genome sequence by the C. elegans Sequencing Consortium is extremely valuable in identifying the conserved genes between C. elegans and C. briggsae.

Antika TR et al. (2023) J Biol Chem "Sequence-specific targeting of C. elegans C-Ala to the D-loop of tRNAAla."

Horng JC, Hsu HL, Nazilah KR, Wang CC, Wang TL, Wang SC, Antika TR, Chuang TH, Chrestella DJ, Wang SW, Tseng YK, Pan HC

[J Biol Chem, 2023]

Alanyl-tRNA synthetase (AlaRS) retains a conserved prototype structure throughout its biology. Nevertheless, its C-terminal domain (C-Ala) is highly diverged and has been shown to play a role in either tRNA or DNA binding. Interestingly, we discovered that Caenorhabditis elegans cytoplasmic C-Ala (Ce-C-Ala_c) robustly binds both ligands. How Ce-C-Ala_c targets its cognate tRNA and whether a similar feature is conserved in its mitochondrial counterpart remain elusive. We show that the N- and C-terminal subdomains of Ce-C-Ala_c are responsible for DNA and tRNA binding, respectively. Ce-C-Ala_c specifically recognized the conserved invariant base G¹⁸ in the D-loop of tRNA^Ala through a highly conserved lysine residue, K934. Despite bearing little resemblance to other C-Ala domains, C. elegans mitochondrial C-Ala (Ce-C-Ala_m) robustly bound both tRNA^Ala and DNA and maintained targeting specificity for the D-loop of its cognate tRNA. This study uncovers the underlying mechanism of how C. elegans C-Ala specifically targets the D-loop of tRNA^Ala.