SF2/ASF regulates proteomic diversity by affecting the balance between translation initiation mechanisms

Abstract

Post-splicing activities have been described for a subset of shuttling serine/arginine-rich splicing regulatory proteins, among them SF2/ASF. We showed that growth factors activate a Ras-PI 3-kinase-Akt/PKB signaling pathway that not only modifies alternative splicing of the fibronectin EDA exon, but also alters in vivo translation of reporter mRNAs containing the EDA binding motif for SF2/ASF, providing two co-regulated levels of isoform-specific amplification. Translation of most eukaryotic mRNAs is initiated via the scanning mechanism, which implicates recognition of the m7G cap at the mRNA 5'-terminus by the eIF4F protein complex. Several viral and cellular mRNAs are translated in a cap-independent manner by the action of cis-acting mRNA elements named internal ribosome entry sites that direct internal ribosome binding to the mRNA. Here we use bicistronic reporters that generate mRNAs carrying two open reading frames, one translated in a cap-dependent manner while the other by internal ribosome entry site-dependent initiation, to show that in vivo over-expression of SF2/ASF increases the ratio between cap-dependent and internal ribosome entry site-dependent translation. Consistently, knocking-down of SF2/ASF causes the opposite effect. Changes in expression levels of SF2/ASF also affect alternative translation of an endogenous mRNA, that one coding for fibroblast growth factor-2. These results strongly suggest a role for SF2/ASF as a regulator of alternative translation, meaning the generation of different proteins by the balance among these two translation initiation mechanisms, and expand the known potential of SF2/ASF to regulate proteomic diversity to the translation field.

Body weight-dependent troponin T alternative splicing is evolutionarily conserved from insects to mammals and is partially impaired in skeletal muscle of obese rats

“…It is becoming apparent that many alternative splicing events require the activity of the same signaling pathways (Lynch, 2007). For example, the phosphatidylinositol 3-kinase (PI3K)/Akt/mTOR (mammalian target of rapamycin) signaling cascade mediates skeletal muscle protein synthesis in response to nutritional and mechanical stimuli (Anthony et al, 2000;Bolster et al, 2003;Kimball and Jefferson, 2006;Kimball et al, 2000;Sasai et al, 2010), and also activates and interacts with members of the serine/arginine (SR)-rich protein family (Blaustein et al, 2009;Karni et al, 2008;Lynch, 2007), a ubiquitous set of alternative splicing factors that affect exon inclusion/exclusion and facilitate translation of specific mRNAs (Blaustein et al, 2005;Sanford et al, 2005). Modulation of sarcomere gene alternative splicing by nutritional and/or mechanically induced signaling through PI3K/Akt/mTOR would add important functionality to the already complex nature of this pathway, and may mediate the dietary and weight-loading effects on troponin T alternative splicing observed in both insects and mammals.…”

Modulation of the Akt Pathway Reveals a Novel Link with PERK/eIF2α, which Is Relevant during Hypoxia

“…Protein extract preparation and western blot analysis were performed as previously described [85]. Primary antibodies used against PERK, peIF2α S51, eIF2α, pAkt S473, Akt, pGSK3β S9, HA, pAkt substrate, PARP and caspase 3 (all from Cell Signaling Technology), GSK3β (clone H76, Santa Cruz), FLAG and actin (1/10000) (Sigma), all were used at 1/1000 at least that something else were indicated.…”

The serine/arginine-rich protein SF2/ASF regulates protein sumoylation

“…However, under this hypothesis, depletion of SF2/ASF by siRNA should reduce PIAS protein levels, which does not seem to be the case. Keeping in mind the already described activity of SF2/ASF in the translation process (5,11,36,37), this potential additional level of control remains to be explored. Considering that SF2/ASF has no SUMO E2 activity, its role as a coregulator of a SUMO E3 ligase adds a further level of regulation to the sumoylation pathway, resembling the case of ubiquitin E3 ligase complexes (31).…”

“…Considering that (i) splicing regulators and ubiquitin/Ubl regulators are found together in protein complexes and share a localization pattern within the cell (32)(33)(34)(35), and (ii) sumoylation regulates different processes that are interconnected with splicing, we decided to gain insight into the role of SR proteins in the SUMO pathway. We chose the best-characterized SR protein, SF2/ASF, whose involvement in signal-induced splicing and translation regulation was previously studied in our group (36,37). Coexpression of SF2/ASF with mature SUMO1 in HEK 293T cells strongly stimulated global protein sumoylation.…”