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Cotranslational signal independent SRP preloading during membrane targeting

View Article: PubMed Central - PubMed

ABSTRACT

Ribosome-associated factors must faithfully decode the limited information available in nascent polypeptides to direct them to their correct cellular fate1. It is unclear how the low complexity information exposed by the nascent chain suffices for accurate recognition by the many factors competing for the limited surface near the ribosomal exit site2,3. Questions remain even for the well-studied cotranslational targeting cycle to the endoplasmic reticulum (ER), involving recognition of linear hydrophobic Signal Sequences (SS) or Transmembrane Domains (TMD) by the Signal Recognition Particle (SRP)4,5. Intriguingly, SRP is in low abundance relative to the large number of ribosome nascent chain complexes (RNCs), yet it accurately selects those destined to the ER6. Despite their overlapping specificities, SRP and the cotranslational Hsp70 SSB display exquisite mutually exclusive selectivity in vivo for their cognate RNCs7,8. To understand cotranslational nascent chain recognition in vivo, we interrogated the cotranslational membrane targeting cycle using ribosome profiling (herein Ribo-seq)9 coupled with biochemical fractionation of ribosome populations. Unexpectedly, SRP preferentially binds secretory RNCs before targeting signals are translated. We show non-coding mRNA elements can promote this signal-independent SRP pre-recruitment. Our study defines the complex kinetic interplay between elongation and determinants in the polypeptide and mRNA modulating SRP-substrate selection and membrane targeting.

No MeSH data available.


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Distinct mechanisms of SRP recruitmenta, Recruitment of SRP to RNCs is expected to increase ribosome-protected reads from SRP-bound monosomes when an SS or TMD is exposed to the cytosol (orange). b, Distributions of SRP-bound ribosome reads on representative transcripts from cycloheximide-treated cultures. Selected transcripts are RCR2, and VBA4. c, The majority of secretory proteins demonstrated SRP enrichment prior to signal exposure. d, Metagene plot of the median value of enrichment of SRP-bound monosomes over polysomes. Included transcripts encode TMDs at least 40 codons from the start codon. Shaded areas represent enrichment before the TMD is encoded (cyan), while the TMD is in the ribosome exit tunnel (lavender), and after the TMD is exposed (orange). e, Two mechanisms for SRP to select secretory mRNA. f,PMP1 and PMP2 were the only TA proteins that enriched SRP. g, The GFP ORF was fused to the indicated 3′ UTRs and expressed in vivo. Srp72p-TAP was immunoprecipitated from the total soluble fraction and RNAs were subject to qPCR. **p ≤ 0.01, n = 3 biological replicates, Welch's t-test. h, Puromycin treatment of lysate from yeast expressing GFP with the PMP1 3′ UTR was followed by SRP immunoprecipitation and qPCR. *p ≤ 0.05, n = 3 biological replicates, Welch's t-test.
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Figure 3: Distinct mechanisms of SRP recruitmenta, Recruitment of SRP to RNCs is expected to increase ribosome-protected reads from SRP-bound monosomes when an SS or TMD is exposed to the cytosol (orange). b, Distributions of SRP-bound ribosome reads on representative transcripts from cycloheximide-treated cultures. Selected transcripts are RCR2, and VBA4. c, The majority of secretory proteins demonstrated SRP enrichment prior to signal exposure. d, Metagene plot of the median value of enrichment of SRP-bound monosomes over polysomes. Included transcripts encode TMDs at least 40 codons from the start codon. Shaded areas represent enrichment before the TMD is encoded (cyan), while the TMD is in the ribosome exit tunnel (lavender), and after the TMD is exposed (orange). e, Two mechanisms for SRP to select secretory mRNA. f,PMP1 and PMP2 were the only TA proteins that enriched SRP. g, The GFP ORF was fused to the indicated 3′ UTRs and expressed in vivo. Srp72p-TAP was immunoprecipitated from the total soluble fraction and RNAs were subject to qPCR. **p ≤ 0.01, n = 3 biological replicates, Welch's t-test. h, Puromycin treatment of lysate from yeast expressing GFP with the PMP1 3′ UTR was followed by SRP immunoprecipitation and qPCR. *p ≤ 0.05, n = 3 biological replicates, Welch's t-test.

Mentions: The canonical model that SRP recognizes the nascent chain after the targeting signal exits the ribosome4 (Fig. 3a) makes several predictions. First, there should be few monosome-protected reads relative to polysomes prior to the first SS/TMD emerging from the ribosome tunnel; second, ribosome footprints should increase beginning approximately 40 codons after the first codon in the targeting signal, and third, monosome reads should decrease after full exposure of the SS/TMD, as SRP-RNCs are delivered to the membrane. Indeed, these patterns were observed in a subset of secretory transcripts with significantly more hydrophobic signals (Fig. 3b, Extended Data Fig. 5c, 2e, f). SRP recruitment to these RNCs only occurred when the translated signals were fully exposed, and not while still in the exit tunnel22,23 (Extended Data Fig. 5d and Supplementary Discussion).


Cotranslational signal independent SRP preloading during membrane targeting
Distinct mechanisms of SRP recruitmenta, Recruitment of SRP to RNCs is expected to increase ribosome-protected reads from SRP-bound monosomes when an SS or TMD is exposed to the cytosol (orange). b, Distributions of SRP-bound ribosome reads on representative transcripts from cycloheximide-treated cultures. Selected transcripts are RCR2, and VBA4. c, The majority of secretory proteins demonstrated SRP enrichment prior to signal exposure. d, Metagene plot of the median value of enrichment of SRP-bound monosomes over polysomes. Included transcripts encode TMDs at least 40 codons from the start codon. Shaded areas represent enrichment before the TMD is encoded (cyan), while the TMD is in the ribosome exit tunnel (lavender), and after the TMD is exposed (orange). e, Two mechanisms for SRP to select secretory mRNA. f,PMP1 and PMP2 were the only TA proteins that enriched SRP. g, The GFP ORF was fused to the indicated 3′ UTRs and expressed in vivo. Srp72p-TAP was immunoprecipitated from the total soluble fraction and RNAs were subject to qPCR. **p ≤ 0.01, n = 3 biological replicates, Welch's t-test. h, Puromycin treatment of lysate from yeast expressing GFP with the PMP1 3′ UTR was followed by SRP immunoprecipitation and qPCR. *p ≤ 0.05, n = 3 biological replicates, Welch's t-test.
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Related In: Results  -  Collection

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Figure 3: Distinct mechanisms of SRP recruitmenta, Recruitment of SRP to RNCs is expected to increase ribosome-protected reads from SRP-bound monosomes when an SS or TMD is exposed to the cytosol (orange). b, Distributions of SRP-bound ribosome reads on representative transcripts from cycloheximide-treated cultures. Selected transcripts are RCR2, and VBA4. c, The majority of secretory proteins demonstrated SRP enrichment prior to signal exposure. d, Metagene plot of the median value of enrichment of SRP-bound monosomes over polysomes. Included transcripts encode TMDs at least 40 codons from the start codon. Shaded areas represent enrichment before the TMD is encoded (cyan), while the TMD is in the ribosome exit tunnel (lavender), and after the TMD is exposed (orange). e, Two mechanisms for SRP to select secretory mRNA. f,PMP1 and PMP2 were the only TA proteins that enriched SRP. g, The GFP ORF was fused to the indicated 3′ UTRs and expressed in vivo. Srp72p-TAP was immunoprecipitated from the total soluble fraction and RNAs were subject to qPCR. **p ≤ 0.01, n = 3 biological replicates, Welch's t-test. h, Puromycin treatment of lysate from yeast expressing GFP with the PMP1 3′ UTR was followed by SRP immunoprecipitation and qPCR. *p ≤ 0.05, n = 3 biological replicates, Welch's t-test.
Mentions: The canonical model that SRP recognizes the nascent chain after the targeting signal exits the ribosome4 (Fig. 3a) makes several predictions. First, there should be few monosome-protected reads relative to polysomes prior to the first SS/TMD emerging from the ribosome tunnel; second, ribosome footprints should increase beginning approximately 40 codons after the first codon in the targeting signal, and third, monosome reads should decrease after full exposure of the SS/TMD, as SRP-RNCs are delivered to the membrane. Indeed, these patterns were observed in a subset of secretory transcripts with significantly more hydrophobic signals (Fig. 3b, Extended Data Fig. 5c, 2e, f). SRP recruitment to these RNCs only occurred when the translated signals were fully exposed, and not while still in the exit tunnel22,23 (Extended Data Fig. 5d and Supplementary Discussion).

View Article: PubMed Central - PubMed

ABSTRACT

Ribosome-associated factors must faithfully decode the limited information available in nascent polypeptides to direct them to their correct cellular fate1. It is unclear how the low complexity information exposed by the nascent chain suffices for accurate recognition by the many factors competing for the limited surface near the ribosomal exit site2,3. Questions remain even for the well-studied cotranslational targeting cycle to the endoplasmic reticulum (ER), involving recognition of linear hydrophobic Signal Sequences (SS) or Transmembrane Domains (TMD) by the Signal Recognition Particle (SRP)4,5. Intriguingly, SRP is in low abundance relative to the large number of ribosome nascent chain complexes (RNCs), yet it accurately selects those destined to the ER6. Despite their overlapping specificities, SRP and the cotranslational Hsp70 SSB display exquisite mutually exclusive selectivity in vivo for their cognate RNCs7,8. To understand cotranslational nascent chain recognition in vivo, we interrogated the cotranslational membrane targeting cycle using ribosome profiling (herein Ribo-seq)9 coupled with biochemical fractionation of ribosome populations. Unexpectedly, SRP preferentially binds secretory RNCs before targeting signals are translated. We show non-coding mRNA elements can promote this signal-independent SRP pre-recruitment. Our study defines the complex kinetic interplay between elongation and determinants in the polypeptide and mRNA modulating SRP-substrate selection and membrane targeting.

No MeSH data available.


Related in: MedlinePlus