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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

Cotranslational enrichment of SRPa, Enrichment of ribosome-protected mRNA reads in the soluble SRP-bound polysome fractions over the total soluble polysome fractions from two biological replicates. b, The number of codons remaining after encoding of first SS or TMD residue, and the corresponding SRP and membrane enrichment scores per ORF. Scores are determined from cultures harvested without added cycloheximide. Enrichment scores are indicated with filled dots, and the scores from the same transcript are linked with a gray line. The vertical dashed line indicates 50 codons, the boundary for TA proteins. Here, only SS that bind Ssh1p directly upon exposure from the RNC are shown. c, Secretory transcripts were classified into two groups based on the ribosome protected-read distributions from SRP-bound polysomes. Some displayed a pronounced increase of reads at positions coincident with to the initial exposure of and SS or TMD by the ribosome, while others did not. Shown here are metagene analysis plots of soluble polysome protected reads from the categorized TMD proteins. For each ORF, the reads at each codon position were divided by the mean reads per codon within the range +20 to +40 after first signal codon. The first 30 codons of each ORF are excluded to avoid the characteristic low-density region near the start codon. The lavender line indicates when the first TMD begins to emerge from the exit tunnel, and the dashed line indicates the position of the read peak. Notably, the total soluble polysome reads depleted in a similar manner for both classes, a read increase was not observed in the total soluble reads, and reads from the SRP-bound transcripts with a peak did not deplete faster than the total soluble reads. These features are consistent with a model where SRP is recruited at the peak site, and elongation then proceeds at the same rate. d, The number of codons remaining after encoding of first SS or TMD and corresponding SRP enrichment. Transcripts are classified by the presence or absence of a read increase following signal exposure, as in c. Note that for SRP-enriched transcripts with signals closest to the terminus (<100 codons), evidence of direct binding between SRP and the nascent chain was always observed. SRP can therefore bind late transmembrane domains immediately after they become exposed by the ribosome. e, Maximum hydrophobicity across targeting signals using an 8-residue averaging window. Only signals with peaks that could be unambiguously attributed to a targeting signal were included. Hydrophobicity was determined by attributing the biological hydrophobicity score to each encoded amino acid54. ***p ≤ 0.001, Wilcoxon rank-sum test. f, Distribution of the distance between the first codon of a targeting signal and the position of the downstream read increase. Only transcripts wherein the increase can be unambiguously attributed to a specific targeting signal were included.
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Figure 6: Cotranslational enrichment of SRPa, Enrichment of ribosome-protected mRNA reads in the soluble SRP-bound polysome fractions over the total soluble polysome fractions from two biological replicates. b, The number of codons remaining after encoding of first SS or TMD residue, and the corresponding SRP and membrane enrichment scores per ORF. Scores are determined from cultures harvested without added cycloheximide. Enrichment scores are indicated with filled dots, and the scores from the same transcript are linked with a gray line. The vertical dashed line indicates 50 codons, the boundary for TA proteins. Here, only SS that bind Ssh1p directly upon exposure from the RNC are shown. c, Secretory transcripts were classified into two groups based on the ribosome protected-read distributions from SRP-bound polysomes. Some displayed a pronounced increase of reads at positions coincident with to the initial exposure of and SS or TMD by the ribosome, while others did not. Shown here are metagene analysis plots of soluble polysome protected reads from the categorized TMD proteins. For each ORF, the reads at each codon position were divided by the mean reads per codon within the range +20 to +40 after first signal codon. The first 30 codons of each ORF are excluded to avoid the characteristic low-density region near the start codon. The lavender line indicates when the first TMD begins to emerge from the exit tunnel, and the dashed line indicates the position of the read peak. Notably, the total soluble polysome reads depleted in a similar manner for both classes, a read increase was not observed in the total soluble reads, and reads from the SRP-bound transcripts with a peak did not deplete faster than the total soluble reads. These features are consistent with a model where SRP is recruited at the peak site, and elongation then proceeds at the same rate. d, The number of codons remaining after encoding of first SS or TMD and corresponding SRP enrichment. Transcripts are classified by the presence or absence of a read increase following signal exposure, as in c. Note that for SRP-enriched transcripts with signals closest to the terminus (<100 codons), evidence of direct binding between SRP and the nascent chain was always observed. SRP can therefore bind late transmembrane domains immediately after they become exposed by the ribosome. e, Maximum hydrophobicity across targeting signals using an 8-residue averaging window. Only signals with peaks that could be unambiguously attributed to a targeting signal were included. Hydrophobicity was determined by attributing the biological hydrophobicity score to each encoded amino acid54. ***p ≤ 0.001, Wilcoxon rank-sum test. f, Distribution of the distance between the first codon of a targeting signal and the position of the downstream read increase. Only transcripts wherein the increase can be unambiguously attributed to a specific targeting signal were included.

Mentions: We next determined which RNCs are substrates of SRP in vivo. Immunoprecipitation of Srp72p from total soluble RNCs was followed by ribosome profiling of both SRP-associated polysomes and monosomes (Fig. 2a and Extended Data Fig. 2a). Few transcripts encoding cytonuclear or mitochondrial proteins were enriched on SRP, confirming its specificity towards ER-destined transcripts. Strikingly, SRP bound to all secretory RNCs that were cotranslationally targeted to the membrane, including SRP-dependent and SRP-independent proteins (Fig. 2b, c).


Cotranslational signal independent SRP preloading during membrane targeting
Cotranslational enrichment of SRPa, Enrichment of ribosome-protected mRNA reads in the soluble SRP-bound polysome fractions over the total soluble polysome fractions from two biological replicates. b, The number of codons remaining after encoding of first SS or TMD residue, and the corresponding SRP and membrane enrichment scores per ORF. Scores are determined from cultures harvested without added cycloheximide. Enrichment scores are indicated with filled dots, and the scores from the same transcript are linked with a gray line. The vertical dashed line indicates 50 codons, the boundary for TA proteins. Here, only SS that bind Ssh1p directly upon exposure from the RNC are shown. c, Secretory transcripts were classified into two groups based on the ribosome protected-read distributions from SRP-bound polysomes. Some displayed a pronounced increase of reads at positions coincident with to the initial exposure of and SS or TMD by the ribosome, while others did not. Shown here are metagene analysis plots of soluble polysome protected reads from the categorized TMD proteins. For each ORF, the reads at each codon position were divided by the mean reads per codon within the range +20 to +40 after first signal codon. The first 30 codons of each ORF are excluded to avoid the characteristic low-density region near the start codon. The lavender line indicates when the first TMD begins to emerge from the exit tunnel, and the dashed line indicates the position of the read peak. Notably, the total soluble polysome reads depleted in a similar manner for both classes, a read increase was not observed in the total soluble reads, and reads from the SRP-bound transcripts with a peak did not deplete faster than the total soluble reads. These features are consistent with a model where SRP is recruited at the peak site, and elongation then proceeds at the same rate. d, The number of codons remaining after encoding of first SS or TMD and corresponding SRP enrichment. Transcripts are classified by the presence or absence of a read increase following signal exposure, as in c. Note that for SRP-enriched transcripts with signals closest to the terminus (<100 codons), evidence of direct binding between SRP and the nascent chain was always observed. SRP can therefore bind late transmembrane domains immediately after they become exposed by the ribosome. e, Maximum hydrophobicity across targeting signals using an 8-residue averaging window. Only signals with peaks that could be unambiguously attributed to a targeting signal were included. Hydrophobicity was determined by attributing the biological hydrophobicity score to each encoded amino acid54. ***p ≤ 0.001, Wilcoxon rank-sum test. f, Distribution of the distance between the first codon of a targeting signal and the position of the downstream read increase. Only transcripts wherein the increase can be unambiguously attributed to a specific targeting signal were included.
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Related In: Results  -  Collection

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Figure 6: Cotranslational enrichment of SRPa, Enrichment of ribosome-protected mRNA reads in the soluble SRP-bound polysome fractions over the total soluble polysome fractions from two biological replicates. b, The number of codons remaining after encoding of first SS or TMD residue, and the corresponding SRP and membrane enrichment scores per ORF. Scores are determined from cultures harvested without added cycloheximide. Enrichment scores are indicated with filled dots, and the scores from the same transcript are linked with a gray line. The vertical dashed line indicates 50 codons, the boundary for TA proteins. Here, only SS that bind Ssh1p directly upon exposure from the RNC are shown. c, Secretory transcripts were classified into two groups based on the ribosome protected-read distributions from SRP-bound polysomes. Some displayed a pronounced increase of reads at positions coincident with to the initial exposure of and SS or TMD by the ribosome, while others did not. Shown here are metagene analysis plots of soluble polysome protected reads from the categorized TMD proteins. For each ORF, the reads at each codon position were divided by the mean reads per codon within the range +20 to +40 after first signal codon. The first 30 codons of each ORF are excluded to avoid the characteristic low-density region near the start codon. The lavender line indicates when the first TMD begins to emerge from the exit tunnel, and the dashed line indicates the position of the read peak. Notably, the total soluble polysome reads depleted in a similar manner for both classes, a read increase was not observed in the total soluble reads, and reads from the SRP-bound transcripts with a peak did not deplete faster than the total soluble reads. These features are consistent with a model where SRP is recruited at the peak site, and elongation then proceeds at the same rate. d, The number of codons remaining after encoding of first SS or TMD and corresponding SRP enrichment. Transcripts are classified by the presence or absence of a read increase following signal exposure, as in c. Note that for SRP-enriched transcripts with signals closest to the terminus (<100 codons), evidence of direct binding between SRP and the nascent chain was always observed. SRP can therefore bind late transmembrane domains immediately after they become exposed by the ribosome. e, Maximum hydrophobicity across targeting signals using an 8-residue averaging window. Only signals with peaks that could be unambiguously attributed to a targeting signal were included. Hydrophobicity was determined by attributing the biological hydrophobicity score to each encoded amino acid54. ***p ≤ 0.001, Wilcoxon rank-sum test. f, Distribution of the distance between the first codon of a targeting signal and the position of the downstream read increase. Only transcripts wherein the increase can be unambiguously attributed to a specific targeting signal were included.
Mentions: We next determined which RNCs are substrates of SRP in vivo. Immunoprecipitation of Srp72p from total soluble RNCs was followed by ribosome profiling of both SRP-associated polysomes and monosomes (Fig. 2a and Extended Data Fig. 2a). Few transcripts encoding cytonuclear or mitochondrial proteins were enriched on SRP, confirming its specificity towards ER-destined transcripts. Strikingly, SRP bound to all secretory RNCs that were cotranslationally targeted to the membrane, including SRP-dependent and SRP-independent proteins (Fig. 2b, c).

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