Seminar in Biological Mechanisms of Aging and Cancer/Journal Club
In the Seminar of Biological Mechanisms of Aging class we discussed the manuscript entitled Splicing factor 1 modulates dietary restriction and TORC1 pathway longevity in C. elegans (Heintz, 2016). Below you will find a graphical abstract and summaries of each figure presented in the manuscript.
Figure 1 b-d
Figure 1 (b-d) highlights the fact that the splicing of the ret-1 reporter changes with age, showing decreased exon 5 inclusion with increasing age. Fig. 1 (b) shows tissue-specific ret-1 splicing in day one C. elegans. The body wall/muscle, hypodermis, and some neurons appear red, which suggests exon 5 exclusion, the intestine appears green, which suggests exon 5 inclusion, and other neurons appear yellow, which show both inclusion and exclusion. The inset of this figure is a control of the same ret-1 splicing reporter without the frameshifts, which explains the complete yellow color. Fig. 1 (c) shows ret-1 splicing at day one and day five. Slightly more exon 5 exclusion is observed at day 5, but a fair amount of variability is observed at this stage as seen in parts c and d. Fig. 1 (d) shows ret-1 splicing at days one, five, and seven. Decrease in green appearance suggests that exon 5 exclusion increases with age. As a control, extended figure 1 (d-o) demonstrates that interfering with spliceosome components deregulates splicing of the ret-1 reporter. Extended data figure 2 (a-b) supports the results found in extended data figure 1, which shows that knockdown of hrp-2 deregulates splicing of exon 5 of the ret-1 gene. Extended data figure 2 (a) shows RNA-seq coverage tracks of ret-1 splicing in control and hrp-2 knockdown samples. Exon 5 was included more frequently in the control and excluded more frequently with hrp-2 knockdown. Extended data figure 2 (b) shows RT-PCR of wild type and hrp-2 RNAi worms in regard to exon 5 splicing of the ret-1 gene. The control shows brighter, larger bands, which indicates that exon 5 was included more frequently, producing longer RNA sequences.
Figure 1 e-g
Figure 1 e C. elegans were placed into two groups, Ad Libitum and Dietary Restriction, to monitor the effects of dietary restriction on splicing patterns. On day 7, the DR C. elegans expressed mostly EGFP. This means, that the worms under the dietary restriction have a youthful splicing pattern, which is characterized by lower exon 5 skipping, resulting in expression of the EGFP tag. Additionally, the aging process in the DR worms was homogenous. The C. elegans that were feed AL expressed mostly mCherry demonstrating aged splicing pattern despite being of the same chronological age as its DR counterpart. This is further supported by ED2-i, which quantified the amount of mCherry and EGFP expressed in AL and DR C. elegans. The amount between the AL and DR were drastically different and heterogenous, therefore there is a definite correlation between C. elegans diet constraints, splicing pattern and preservation of splicing quality in organisms.
Figure 1 f-g The C.elegans that were fed ab libitum demonstrated heterogeneous splicing patterns and differences at rate of aging. In order to determine if the differences in rate of aging were due to differences in the splicing pattern, on day 6 of their life, C. elegans were sorted into two groups, A and B, based on their splicing patterns. As shown in figure 1f, group A consisted of C. elegans that expressed mostly mCherry, which had similar behavior to C. elegans that were under dietary restriction. Group B consisted of C. elegans that expressed mostly EGFP, which was characteristic of C. elegans that fed AL.
Figure 1 h-i Figure 1h shows the relationship between sfa-1 RNAi and the dietary restricted feeding plan (DR). It can be seen that when C. elegans are on the DR, their average lifespan is much longer than the wild-type (WT) C. elegans, which were fed a normal AL feeding plan. When the DR worms had the sfa-1 RNAi, their average lifespan was significantly decreased, similar to the lifespan of the WT worms. When the WT worms had the sfa-1 RNAi, these worms, on average, lived slightly longer than when they didn’t have the sfa-1 RNAi. A similar analysis of the relationships of various spliceosome factors is shown in Extended Data Fig. 2k-p. Some of the factors such as uaf-2, hrp-2 and snr-1 did affect the lifespan of the C. elegans but some did not which leads to the conclusion that not all spliceosome factors contribute or decline lifespan. A further study of the uaf-2 and the sfa-1 factors can be explained by Extended Data Fig. 3a. Although, both genes for these factors are expressed on the same operon, sfa-1 RNAi had no effect on the expression of uaf-2. Figure 1i is a PCR analysis of the splicing pattern for the tos-1 gene and indicates that this gene requires SFA-1.The bottom bands on this gel analysis indicate that they are from protein without exon 5. The lanes with wild type (WT) day 15 (both control and sfa-1 RNAi) and eat-2(ad1116) (with sfa-1 RNAi) at day 15 have the second band indicating effects of aging. C. elgans with the eat-2(ad1116) do not have as dark of bands at day 15. In Extended Data Figure 3 b and c pumping rates of the control and the eat-2(ad1116) model are compared to see if the sfa-1 RNAi has an effect on eating of the C. elegans. It is seen that there is no effect in day 1 or day 4 of adulthood. In the Extended Data Figure 3 e PCR is used to analyze the ret-1 gene splicing patterns of the wildtype and dietary restriction animals. This figure indicates that the sfa-1 RNAi has a similar effect to the effect it had with the tos-1 gene. This effect is also seen in Extended Data Figure 3 d where the dietary restriction animals with sfa-1 RNAi shows signs of aging on day 7.
Figure 2 primarily conveys the relationship between dietary regime and the frequency of splicing mistakes over time. Figure 2a and 2b illustrate that as the worms increase in chronological age, their splicing efficiency declines. The green columns indicate a lot less un-annotated junctions and introns reads, as compared to the red and orange columns, which convey a greater percentage of the splicing noise occurring at a greater chronological age of C. elegans. In addition, figure 2c and 2d seek to highlight the role of sfa-1 in dietary restricted worms, by comparing dietary restricted worms with and without the sfa-1 knockdown. It is clear that dietary restriction coupled with sfa-1 over expression leads to greater extension of lifespan, as well as delaying the onset of splicing defects. Figure 2d illustrates that sfa-1 is essential to the extension of lifespan and repression of splicing defects through the process of dietary restriction.
The data presented in figure 3 define the effects of dietary restriction that are specifically associated with SFA-1 using several experimental techniques. The data in figure 3a represent differential gene expression associated with KEGG pathways between animals that were fed ad libitium, animals on dietary restriction, and animals on dietary restriction (DR) plus sfa-1 RNAi (DR SFA). The green rectangle in the upper right of this figure highlights the change in gene expression between DR and DR SFA in the KEGG pathways associated with lipid metabolism. Specifically, animals under DR have an upregulation (red) of genes associated with lipid metabolism and this upregulation is SFA-1 dependent because the DR SFA animals have a downregulation of these genes (blue). Figure 3b shows the relationship between genes showing intron inclusion with age that are functionally enriched for metabolic processes. There are more genes associated with metabolic processes in the AL and DR SFA animals as compared to the DR animals, which is consisted with the previous data suggesting that the effects of DR are dependent on SFA-1. Figures 3c and 3d confirm these findings by demonstrating that SFA-1 mediates the effects of DR directly. In figure 3c, animals fed AL and AL SFA have a lower maximal respiratory capacity with age, which is attenuated by animals under DR. In figure 3d they use a GFP tagged reporter strain of C. elegans to determine that animals under DR have high expression (more green) of acyl-CoA synthase, which is necessary for fatty acid oxidation during starvation, as compared to animals under DR without SFA-1 (less green). Overall these data suggest that SFA-1 has a direct role in modulating metabolism and the effects of DR in C. elegans.
The authors aimed this part of the study at identifying the mechanisms that link dietary restrictions to a longer life span. They compared the effects of three known mutations in genes on the longevity of an organism whose diet is restricted: aak-2, daf-16, and raga-1. Notice that in pictures (a) and (b) there is relatively the same amount of green in dietary restricted wild-type and the two dietary restricted mutants aak-2 and daf-2. This indicates that the mutations aak-2 and dat-16 do not disrupt the benefits that dietary restriction has on splicing of ret-1 in C. elengans. The opposite is seen with the raga-1 mutation in picture (c). There is no green in the dietary restricted raga-1 mutant, this indicates that the raga-1 mutation abolishes the benefits of dietary restriction on increasing longevity. Overall, it can be seen in these three pictures that AAK-2 and DAF-2 don’t affect the splicing of dietary restricted C.elegans, but RAGA-1 is needed for the effect of dietary restriction on the splicing of ret-1. In order to understand the role of SFA-1 in the increase of lifespan in C. elegans, the experiments tested for the impact of sfa-1 RNA interference (RNAi) on the average lifespan of a population of C. elegans. As seen in graph (d), the dashed purple line shows that sfa-1 RNAi fully suppresses lifespan increase by nullifying the gene raga-1. The same pattern is show in graph e, which shows a dashed green line to represent the combination of sfa-1 RNAi and the raga-1 gene. sfa-1 fully suppresses lifespan increase by nullifying the gene rsks-1 (graph e). Graph F shows that overexpression of the gene sfa-1, lines blue and red, alone is able to result in an increase in lifespan of the C. elegans. Diagram g is a model that shows that the suppression of the TORC1 pathway, either by dietary restriction or AMPK, is needed in order to SFA-1 gene to be able to result in longevity in C. elegans.