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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.

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Cotranslational membrane enrichmenta, Distributions of the open reading frame (ORF) enrichment of ribosome-protected reads in the membrane fraction over the soluble fraction. ORFs were alternatively classified by expected SRP dependence11. **p ≤ 0.01, Wilcoxon rank-sum test. b, Ribosome-protected reads at each codon of an example transmembrane protein OLE1. Membrane topology is indicated above, with the first TMD in lavender. c, Metagene analysis of soluble fraction polysome-protected reads from transcripts that were at least 2-fold membrane enriched. ORFs were aligned at the targeting signal and scaled. d, Cotranslational membrane targeting is in competition with elongation. e, Elongation inhibitors provide additional time for polysomes exposing a targeting signal to localize to the membrane. f, Membrane enrichment was limited by the length of the reading frame remaining after the encoding of targeting signals. The vertical dashed line indicates 50 codons.
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Figure 1: Cotranslational membrane enrichmenta, Distributions of the open reading frame (ORF) enrichment of ribosome-protected reads in the membrane fraction over the soluble fraction. ORFs were alternatively classified by expected SRP dependence11. **p ≤ 0.01, Wilcoxon rank-sum test. b, Ribosome-protected reads at each codon of an example transmembrane protein OLE1. Membrane topology is indicated above, with the first TMD in lavender. c, Metagene analysis of soluble fraction polysome-protected reads from transcripts that were at least 2-fold membrane enriched. ORFs were aligned at the targeting signal and scaled. d, Cotranslational membrane targeting is in competition with elongation. e, Elongation inhibitors provide additional time for polysomes exposing a targeting signal to localize to the membrane. f, Membrane enrichment was limited by the length of the reading frame remaining after the encoding of targeting signals. The vertical dashed line indicates 50 codons.

Mentions: Secretory proteins are proposed to target to the ER membrane either co- or post-translationally for subsequent translocation10–12. Mechanistic models of ER targeting and the role of SRP derive primarily from cell-free systems using model proteins10,13, raising the question of how these pathways function in the cell. To investigate membrane targeting in vivo, we fractionated soluble and membrane-attached ribosomes from yeast cells, and then used Ribo-seq to compare the ribosome-protected mRNA footprints from polysomes obtained from both fractions (Extended Data Fig. 1a). We derived a cotranslational membrane enrichment score for each coding sequence (Methods, Extended Data Fig. 1b, Supplementary Table 1). Transcripts encoding cytosolic or nuclear proteins (herein cytonuclear) were preferentially translated on cytosolic ribosomes and not enriched on membrane polysomes (Fig. 1a). Tail-anchored (TA) proteins, whose single TMD at the carboxyl terminus is only revealed posttranslationally14, were also translated on cytosolic ribosomes. In contrast, many nuclear-encoded mitochondrial protein transcripts were enriched in the membrane-bound ribosome fraction, as expected15. Transcripts encoding ER-destined secretory proteins were highly enriched on membrane-bound ribosomes. Proteins containing a SS or TMD had comparable cotranslational membrane enrichment, conflicting with the notion that the targeting signal itself distinguishes which proteins are targeted co- or post-translationally to the ER11,12 (Fig. 1a).


Cotranslational signal independent SRP preloading during membrane targeting
Cotranslational membrane enrichmenta, Distributions of the open reading frame (ORF) enrichment of ribosome-protected reads in the membrane fraction over the soluble fraction. ORFs were alternatively classified by expected SRP dependence11. **p ≤ 0.01, Wilcoxon rank-sum test. b, Ribosome-protected reads at each codon of an example transmembrane protein OLE1. Membrane topology is indicated above, with the first TMD in lavender. c, Metagene analysis of soluble fraction polysome-protected reads from transcripts that were at least 2-fold membrane enriched. ORFs were aligned at the targeting signal and scaled. d, Cotranslational membrane targeting is in competition with elongation. e, Elongation inhibitors provide additional time for polysomes exposing a targeting signal to localize to the membrane. f, Membrane enrichment was limited by the length of the reading frame remaining after the encoding of targeting signals. The vertical dashed line indicates 50 codons.
© Copyright Policy - permission-link
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC5120976&req=5

Figure 1: Cotranslational membrane enrichmenta, Distributions of the open reading frame (ORF) enrichment of ribosome-protected reads in the membrane fraction over the soluble fraction. ORFs were alternatively classified by expected SRP dependence11. **p ≤ 0.01, Wilcoxon rank-sum test. b, Ribosome-protected reads at each codon of an example transmembrane protein OLE1. Membrane topology is indicated above, with the first TMD in lavender. c, Metagene analysis of soluble fraction polysome-protected reads from transcripts that were at least 2-fold membrane enriched. ORFs were aligned at the targeting signal and scaled. d, Cotranslational membrane targeting is in competition with elongation. e, Elongation inhibitors provide additional time for polysomes exposing a targeting signal to localize to the membrane. f, Membrane enrichment was limited by the length of the reading frame remaining after the encoding of targeting signals. The vertical dashed line indicates 50 codons.
Mentions: Secretory proteins are proposed to target to the ER membrane either co- or post-translationally for subsequent translocation10–12. Mechanistic models of ER targeting and the role of SRP derive primarily from cell-free systems using model proteins10,13, raising the question of how these pathways function in the cell. To investigate membrane targeting in vivo, we fractionated soluble and membrane-attached ribosomes from yeast cells, and then used Ribo-seq to compare the ribosome-protected mRNA footprints from polysomes obtained from both fractions (Extended Data Fig. 1a). We derived a cotranslational membrane enrichment score for each coding sequence (Methods, Extended Data Fig. 1b, Supplementary Table 1). Transcripts encoding cytosolic or nuclear proteins (herein cytonuclear) were preferentially translated on cytosolic ribosomes and not enriched on membrane polysomes (Fig. 1a). Tail-anchored (TA) proteins, whose single TMD at the carboxyl terminus is only revealed posttranslationally14, were also translated on cytosolic ribosomes. In contrast, many nuclear-encoded mitochondrial protein transcripts were enriched in the membrane-bound ribosome fraction, as expected15. Transcripts encoding ER-destined secretory proteins were highly enriched on membrane-bound ribosomes. Proteins containing a SS or TMD had comparable cotranslational membrane enrichment, conflicting with the notion that the targeting signal itself distinguishes which proteins are targeted co- or post-translationally to the ER11,12 (Fig. 1a).

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