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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.


Related in: MedlinePlus

The role of the UTR from PMP1 and PMP2a, The cotranslational SRP enrichment of the PMP1 and PMP2 ORFs was similar to other bona fide secretory proteins, such as SEC61. In contrast, cytosolic proteins such as tubulin (TUB2), were not enriched. The enrichment scores are determined from the SRP-bound and total soluble polysomes from two biological replicates harvested without added cycloheximide. b, Distribution of ribosome-protected reads from soluble polysomes within the PMP1 and PMP2 ORFs. c, Membrane enrichment, determined by qPCR, of the mRNA of GFP fused to the indicated 3′ UTRs. The coding sequence of endogenous SEC61 transcript was also amplified as a control for a membrane localized transcript. **p ≤ 0.01, n = 3 biological replicates, Welch's t-test. d, Localization of mature GFP. The scale bar indicates 5 μM. Yeast were grown to mid-log phase and imaged using an Axio Observer Z1 with a Plan-Apochromat 100x/1.4 oil immersion objective (Zeiss). Z-stacks were deconvoluted by the iterative maximum likelihood algorithm in ZEN (Zeiss) and single planes are shown. Images were representative from a set of 2 replicated assays. e, Yeast growth after replacement of the endogenous 3′ UTR of PMP1 with the 3′ UTR of tubulin. Also shown is a complete deletion of PMP1 ORF34. Gibson assembly53 was used to fuse the 300 nt TUB2 3′ UTR to the KlURA3 cassette into SmaI digested pUC19. The TUB2-UTR-URA3 element was PCR amplified, including 40 nt overhangs matching genomic sequences, and replaced the 650 nt immediately following the PMP1 coding sequence in strain BY4741 by homologous recombination. Image is representative from a set of 3 replicated assays. f, Nascent chain independent SRP recognition may require ribosomes. Puromycin treatment of lysates disrupts elongating, but not initiating, ribosomes. g, Transcripts showing only canonical recognition are more sensitive to puromycin. This is consistent with puromycin resistance of SRP that has pre-recruited to initiating ribosomes. h, Membrane enrichment of the GFP-PMP1 construct or SEC61 mRNA after lysates were incubated with puromycin. **p ≤ 0.01, n = 3 biological replicates, Welch's t-test.
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Figure 10: The role of the UTR from PMP1 and PMP2a, The cotranslational SRP enrichment of the PMP1 and PMP2 ORFs was similar to other bona fide secretory proteins, such as SEC61. In contrast, cytosolic proteins such as tubulin (TUB2), were not enriched. The enrichment scores are determined from the SRP-bound and total soluble polysomes from two biological replicates harvested without added cycloheximide. b, Distribution of ribosome-protected reads from soluble polysomes within the PMP1 and PMP2 ORFs. c, Membrane enrichment, determined by qPCR, of the mRNA of GFP fused to the indicated 3′ UTRs. The coding sequence of endogenous SEC61 transcript was also amplified as a control for a membrane localized transcript. **p ≤ 0.01, n = 3 biological replicates, Welch's t-test. d, Localization of mature GFP. The scale bar indicates 5 μM. Yeast were grown to mid-log phase and imaged using an Axio Observer Z1 with a Plan-Apochromat 100x/1.4 oil immersion objective (Zeiss). Z-stacks were deconvoluted by the iterative maximum likelihood algorithm in ZEN (Zeiss) and single planes are shown. Images were representative from a set of 2 replicated assays. e, Yeast growth after replacement of the endogenous 3′ UTR of PMP1 with the 3′ UTR of tubulin. Also shown is a complete deletion of PMP1 ORF34. Gibson assembly53 was used to fuse the 300 nt TUB2 3′ UTR to the KlURA3 cassette into SmaI digested pUC19. The TUB2-UTR-URA3 element was PCR amplified, including 40 nt overhangs matching genomic sequences, and replaced the 650 nt immediately following the PMP1 coding sequence in strain BY4741 by homologous recombination. Image is representative from a set of 3 replicated assays. f, Nascent chain independent SRP recognition may require ribosomes. Puromycin treatment of lysates disrupts elongating, but not initiating, ribosomes. g, Transcripts showing only canonical recognition are more sensitive to puromycin. This is consistent with puromycin resistance of SRP that has pre-recruited to initiating ribosomes. h, Membrane enrichment of the GFP-PMP1 construct or SEC61 mRNA after lysates were incubated with puromycin. **p ≤ 0.01, n = 3 biological replicates, Welch's t-test.

Mentions: To begin to understand the determinants that confer specific recruitment of SRP without an exposed SS or TMD, we examined the most extreme cases of nascent chain independent SRP-recruitment. PMP1 and PMP2 encode two abundant, small membrane proteins of 40 and 42 amino acids, respectively. Even though the entire proteins are smaller than the length of the ribosomal tunnel, PMP1 and PMP2 RNCs bind to SRP throughout translation (Fig. 3f and Extended Data Fig. 6a, b).


Cotranslational signal independent SRP preloading during membrane targeting
The role of the UTR from PMP1 and PMP2a, The cotranslational SRP enrichment of the PMP1 and PMP2 ORFs was similar to other bona fide secretory proteins, such as SEC61. In contrast, cytosolic proteins such as tubulin (TUB2), were not enriched. The enrichment scores are determined from the SRP-bound and total soluble polysomes from two biological replicates harvested without added cycloheximide. b, Distribution of ribosome-protected reads from soluble polysomes within the PMP1 and PMP2 ORFs. c, Membrane enrichment, determined by qPCR, of the mRNA of GFP fused to the indicated 3′ UTRs. The coding sequence of endogenous SEC61 transcript was also amplified as a control for a membrane localized transcript. **p ≤ 0.01, n = 3 biological replicates, Welch's t-test. d, Localization of mature GFP. The scale bar indicates 5 μM. Yeast were grown to mid-log phase and imaged using an Axio Observer Z1 with a Plan-Apochromat 100x/1.4 oil immersion objective (Zeiss). Z-stacks were deconvoluted by the iterative maximum likelihood algorithm in ZEN (Zeiss) and single planes are shown. Images were representative from a set of 2 replicated assays. e, Yeast growth after replacement of the endogenous 3′ UTR of PMP1 with the 3′ UTR of tubulin. Also shown is a complete deletion of PMP1 ORF34. Gibson assembly53 was used to fuse the 300 nt TUB2 3′ UTR to the KlURA3 cassette into SmaI digested pUC19. The TUB2-UTR-URA3 element was PCR amplified, including 40 nt overhangs matching genomic sequences, and replaced the 650 nt immediately following the PMP1 coding sequence in strain BY4741 by homologous recombination. Image is representative from a set of 3 replicated assays. f, Nascent chain independent SRP recognition may require ribosomes. Puromycin treatment of lysates disrupts elongating, but not initiating, ribosomes. g, Transcripts showing only canonical recognition are more sensitive to puromycin. This is consistent with puromycin resistance of SRP that has pre-recruited to initiating ribosomes. h, Membrane enrichment of the GFP-PMP1 construct or SEC61 mRNA after lysates were incubated with puromycin. **p ≤ 0.01, n = 3 biological replicates, Welch's t-test.
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Figure 10: The role of the UTR from PMP1 and PMP2a, The cotranslational SRP enrichment of the PMP1 and PMP2 ORFs was similar to other bona fide secretory proteins, such as SEC61. In contrast, cytosolic proteins such as tubulin (TUB2), were not enriched. The enrichment scores are determined from the SRP-bound and total soluble polysomes from two biological replicates harvested without added cycloheximide. b, Distribution of ribosome-protected reads from soluble polysomes within the PMP1 and PMP2 ORFs. c, Membrane enrichment, determined by qPCR, of the mRNA of GFP fused to the indicated 3′ UTRs. The coding sequence of endogenous SEC61 transcript was also amplified as a control for a membrane localized transcript. **p ≤ 0.01, n = 3 biological replicates, Welch's t-test. d, Localization of mature GFP. The scale bar indicates 5 μM. Yeast were grown to mid-log phase and imaged using an Axio Observer Z1 with a Plan-Apochromat 100x/1.4 oil immersion objective (Zeiss). Z-stacks were deconvoluted by the iterative maximum likelihood algorithm in ZEN (Zeiss) and single planes are shown. Images were representative from a set of 2 replicated assays. e, Yeast growth after replacement of the endogenous 3′ UTR of PMP1 with the 3′ UTR of tubulin. Also shown is a complete deletion of PMP1 ORF34. Gibson assembly53 was used to fuse the 300 nt TUB2 3′ UTR to the KlURA3 cassette into SmaI digested pUC19. The TUB2-UTR-URA3 element was PCR amplified, including 40 nt overhangs matching genomic sequences, and replaced the 650 nt immediately following the PMP1 coding sequence in strain BY4741 by homologous recombination. Image is representative from a set of 3 replicated assays. f, Nascent chain independent SRP recognition may require ribosomes. Puromycin treatment of lysates disrupts elongating, but not initiating, ribosomes. g, Transcripts showing only canonical recognition are more sensitive to puromycin. This is consistent with puromycin resistance of SRP that has pre-recruited to initiating ribosomes. h, Membrane enrichment of the GFP-PMP1 construct or SEC61 mRNA after lysates were incubated with puromycin. **p ≤ 0.01, n = 3 biological replicates, Welch's t-test.
Mentions: To begin to understand the determinants that confer specific recruitment of SRP without an exposed SS or TMD, we examined the most extreme cases of nascent chain independent SRP-recruitment. PMP1 and PMP2 encode two abundant, small membrane proteins of 40 and 42 amino acids, respectively. Even though the entire proteins are smaller than the length of the ribosomal tunnel, PMP1 and PMP2 RNCs bind to SRP throughout translation (Fig. 3f and Extended Data Fig. 6a, b).

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