Systems Biology Analysis of Human Primary T Cells Identifies SAP145 as Rate Limiting for the G₁→S Phase Transition.

We identified a G₀→G₁ commitment point in primary human T cells that controls entry into the cell cycle from quiescence. We demonstrated proof of principle that cellular pathways regulating cell cycle progression and effector functions that normally coincide during CD3/CD28 stimulation can be uncoupled experimentally. We have now used systems biology approaches to identify nuclear protein networks in primary human T cells that are regulated during the transition from quiescence into the cell cycle (G₀→G₁→S-phase). First we sequenced proteins that became bound to chromatin & nuclear matrix in G₁ but were not bound in G₀ and vice versa by mass spectrometry. Bioinformatic analysis identified 76 proteins specifically bound in G₀ not G₁ and 254 bound in G₁ not G₀. 179 of the 254 proteins bound in G₁ not G₀ (i.e. dynamic protein changes) were mapped to the 55,000 human protein interaction dataset. These are involved in numerous cellular functions, including epigenetics, transcription, RNA splicing & transport, and others. Cell cycle regulated chromatin/matrix binding of a subset was verified by western blotting (2/2 bound in G₀ not G₁ and 22/23 bound in G₁ not G₀). One of the proteins induced and bound in G₁ was SAP145 (SF3B2). This is a component of the ubiquitous SF3b RNA splicing complex, involved in both major (U2-type) and minor (U12-type) spliceosomes. Since SAP145 is induced during G₁ we investigated whether there was a role for SAP145 in regulating cell cycle progression. T cells depleted of SAP145 by siRNA enter G₁ from G₀ but progress poorly through S phase and die, probably by apoptosis. The same occurs if another component of the SF3B complex, SAP49 (SF3B4) is depleted with siRNA, indicating that the effect is due to depleting the complex rather than the individual SF3B protein. Proteins that are induced during G₁ by CD3/CD28 stimulation e.g. cyclin D3, Cdc6 and cdc2 are produced normally when SAP145 is depleted, suggesting that their pre-mRNAs are spliced normally. In contrast, the expression of p107 and cyclin A2 are reduced markedly when SAP145 is depleted. Therefore, a systems biology approach to analysing cell cycle transitions identifies the splicing protein, SAP145 as rate-limiting for the G₁ →S phase cell cycle transition but not for the transition from G₀→G₁.