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. Author manuscript; available in PMC: 2015 Jan 16.
Published in final edited form as: Cell. 2014 Jan 16;156(0):146–157. doi: 10.1016/j.cell.2013.12.017

Figure 2. Defects in the Signal Sequence or in a Targeting Factor, SRP, Lead to Decreased Levels of a Secretory Protein mRNA.

Figure 2

(A and B) Effect of deletions in signal sequence on PPL mRNA levels. PPL and control mRNAs were analyzed by Northern blot (A) or qPCR (B; n=9, mean ± SD) 42 h after transfection of HeLa Tet-On with WT and mutated PPL plasmids. (C) Presence of a mutated PPL signal sequence (PPL- Δ4L) in hybrid proteins containing the mature part of a1-antitrypsin (AT) or carbonic anhydrase IV (CA4) is sufficient to trigger mRNA depletion. mRNA levels measured by qPCR (n=3, mean ± SD) are shown relatively to mRNA levels of corresponding hybrid proteins containing WT PPL signal sequence (PPL-WT). (D) Deletions (indicated by dashes) in the natural signal sequences of secretory proteins AT or CA4. (E) Deletions in the hydrophobic core of natural signal sequences of AT or CA4 proteins lead to a decrease in their mRNA levels. Graph shows mRNA levels measured by qPCR (n=3, mean ± SD). (F) Scheme shows differences in biogenesis of secretory and cytosolic proteins. When the signal sequence of a secretory protein emerges from the ribosomal tunnel it is recognized by SRP, and the complex is targeted to SRP receptor and finally to a translocon in ER membrane. Nascent chains of cytosolic proteins do not have signal sequences, however, their nascent chains are recognized by ribosome-associated chaperones and that help them fold in the cytosol. (G–H) SRP depletion causes a reduction in the level of secretory protein mRNA. Detection of PPL, and actin mRNAs (Northern blot), SRP54, PPL and actin proteins (Western blot) in HeLa Tet-On cells transfected with siRNA for SRP54 and WT or Δ4L PPL plasmids as indicated (G). Quantification of WT PPL (open bars) or Δ4L mutant (black bars) mRNAs by qPCR in independent sets of SRP54 knockdown experiments (n=3, mean ± SD) (H). (I) Knockdown of SRP receptor subunits SRα and SRβ, or translocon component, Sec61α, does not affect PPL mRNA level (qPCR, n=3, mean ± SD). WT PPL (open bars), Δ4L mutant (black bars). (J) SRP depletion causes reduction in endogenous mRNA levels of secretory and ER proteins. Measured by qPCR (n=3, mean ± SD) mRNA levels in SRP depleted cells are shown relatively those treated with control siRNA (taken as 1). Intestinal alkaline phosphatase (AP) is a secretory protein, Bip and calreticulin (Calr) are ER lumen proteins. mRNA levels of the cytosolic protein HPRT and the SRP-independent ER membrane-associated protein SRα were not altered. See also Figures S2 and Table S1.