The heterodimeric subunit SRP9/14 of the signal recognition particle functions as permuted single polypeptide chain.

The targeting of nascent polypeptide chains to the endoplasmic reticulum is mediated by a cytoplasmic ribonucleoprotein, the signal recognition particle (SRP). The 9 kD (SRP9) and the 14 kD (SRP14) subunits of SRP are required to confer elongation arrest activity to the particle. SRP9 and SRP14 form a heterodimer which specifically binds to SRP RNA. We have constructed cDNAs that encode single polypeptide chains comprising SRP9 and SRP14 sequences in the two possible permutations linked by a 17 amino acid peptide. We found that both fusion proteins specifically bound to SRP RNA as monomeric molecules folded into a heterodimer-like structure. Our results corroborate the previous hypothesis that the authentic heterodimer binds to SRP RNA in equimolar ratio. In addition, both fusion proteins conferred elongation arrest activity to SRP(-9/14), which lacks this function, and one fusion protein could functionally replace the heterodimer in the translocation assay. Thus, the normal N-and C-termini of both proteins have no essential role in folding, RNA-binding and in mediating the biological activities. The possibility to express the heterodimeric complex as a single polypeptide chain facilitates the analysis of its functions and its structure in vivo and in vitro

The SRP9/14 subunit of the human signal recognition particle binds to a variety of Alu-like RNAs and with higher affinity than its mouse homolog.

The heterodimeric subunit, SRP9/14, of the signal recognition particle (SRP) has previously been found to bind to scAlu and scB1 RNAs in vitro and to exist in large excess over SRP in anthropoid cells. Here we show that human and mouse SRP9/14 bind with high affinities to other Alu-like RNAs of different evolutionary ages including the neuron-specific BC200 RNA. The relative dissociation constants of the different RNA-protein complexes are inversely proportional to the evolutionary distance between the Alu RNA species and 7SL RNA. In addition, the human SRP9/14 binds with higher affinity than mouse SRP9/14 to all RNAs analyzed and this difference is not explained by the additional C-terminal domain present in the anthropoid SRP14. The conservation of high affinity interactions between SRP9/14 and Alu-like RNAs strongly indicates that these Alu-like RNPs exist in vivo and that they have cellular functions. The observation that human SRP9/14 binds better than its mouse counterpart to distantly related Alu RNAs, such as recently transposed elements, suggests that the anthropoid-specific excess of SRP9/14 may have a role in controlling Alu amplification rather than in compensating a defect in SRP assembly and functions

Calpain-mediated cleavage of Atg5 switches autophagy to apoptosis

Autophagy-related gene (Atg) 5 is a gene product required for the formation of autophagosomes. Here, we report that Atg5, in addition to the promotion of autophagy, enhances susceptibility towards apoptotic stimuli. Enforced expression of Atg5-sensitized tumour cells to anticancer drug treatment both in vitro and in vivo. In contrast, silencing the Atg5 gene with short interfering RNA (siRNA) resulted in partial resistance to chemotherapy. Apoptosis was associated with calpain-mediated Atg5 cleavage, resulting in an amino-terminal cleavage product with a relative molecular mass of 24,000 (Mr 24K). Atg5 cleavage was observed independent of the cell type and the apoptotic stimulus, suggesting that calpain activation and Atg5 cleavage are general phenomena in apoptotic cells. Truncated Atg5 translocated from the cytosol to mitochondria, associated with the anti-apoptotic molecule Bcl-xL and triggered cytochrome c release and caspase activation. Taken together, calpain-mediated Atg5 cleavage provokes apoptotic cell death, therefore, represents a molecular link between autophagy and apoptosis--a finding with potential importance for clinical anticancer therapies