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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

Elongation pausing and local SRP recruitmenta, b, Local increases of ribosome-protected reads from membrane-bound polysomes, indicated with orange lines, were coincident with rare codons, as in cell division cycle protein 1 (CDC1, a) or polybasic nascent chains, as in the plasma membrane G-protein coupled receptor (GPR1, b). Soluble SRP-bound polysome-protected reads were further increased at the same positions. c, In these cases, hydrophobic sequences in the nascent chain were exposed to the cytosol at the locations of increased reads, which were coincident with elongation attenuators. d, Translational efficiencies for the 6 codons following, and the number of stalling residues within the 10 residues preceding, the sites of increased SRP-bound ribosome reads. Translational efficiency was determined by attributing the nTE score to each codon55. Residues that were found to stall the ribosome, based on previous investigation20,56,57, were lysine, arginine, glutamate, aspartate, proline and glycine. Because of variation in specific motifs, and uncertainty in whether these motifs are additive, we simply compared the total number of these residues in the indicated 10 residue spans. Sets of 10,000 random sequences, at least 10 amino acids from the stop codon, were sampled from 5907 non-dubious ORFs, and translational efficiency and stalling residues were determined over 6 or 10 codon spans. *p ≤ 0.05, **p ≤ 0.01, Wilcoxon rank-sum tests. e, The targeting signals that recruited SRP directly to the nascent chain unusually far from the encoding of the signal had SRP-binding sites coincident with intrinsic elongation attenuation. Secretory protein transcripts that showed an increase in SRP-bound protected reads (see Extended Data Fig. 2c, f) were further classified by the position of the peak relative to the first signal codon. Transcripts with peaks found at least 80 codons after the signal had significantly lower translational efficiency in the 6 codons following the peak. These transcripts also had a greater, but not statistically significant, amount of stalling amino acids in the 10 residues preceding the peak. *p ≤ 0.05, Wilcoxon rank-sum tests. f, Similar increases in SRP-bound reads were observed for certain non-secretory proteins as exemplified by phosphoacetylglucosamine mutase (PCM1) and tRNASer Um44 2′-O-methyltransferase (TRM44). Hydrophobic sequences in non-secretory proteins, coupled with attenuation of elongation, may lead to SRP recruitment.
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Figure 7: Elongation pausing and local SRP recruitmenta, b, Local increases of ribosome-protected reads from membrane-bound polysomes, indicated with orange lines, were coincident with rare codons, as in cell division cycle protein 1 (CDC1, a) or polybasic nascent chains, as in the plasma membrane G-protein coupled receptor (GPR1, b). Soluble SRP-bound polysome-protected reads were further increased at the same positions. c, In these cases, hydrophobic sequences in the nascent chain were exposed to the cytosol at the locations of increased reads, which were coincident with elongation attenuators. d, Translational efficiencies for the 6 codons following, and the number of stalling residues within the 10 residues preceding, the sites of increased SRP-bound ribosome reads. Translational efficiency was determined by attributing the nTE score to each codon55. Residues that were found to stall the ribosome, based on previous investigation20,56,57, were lysine, arginine, glutamate, aspartate, proline and glycine. Because of variation in specific motifs, and uncertainty in whether these motifs are additive, we simply compared the total number of these residues in the indicated 10 residue spans. Sets of 10,000 random sequences, at least 10 amino acids from the stop codon, were sampled from 5907 non-dubious ORFs, and translational efficiency and stalling residues were determined over 6 or 10 codon spans. *p ≤ 0.05, **p ≤ 0.01, Wilcoxon rank-sum tests. e, The targeting signals that recruited SRP directly to the nascent chain unusually far from the encoding of the signal had SRP-binding sites coincident with intrinsic elongation attenuation. Secretory protein transcripts that showed an increase in SRP-bound protected reads (see Extended Data Fig. 2c, f) were further classified by the position of the peak relative to the first signal codon. Transcripts with peaks found at least 80 codons after the signal had significantly lower translational efficiency in the 6 codons following the peak. These transcripts also had a greater, but not statistically significant, amount of stalling amino acids in the 10 residues preceding the peak. *p ≤ 0.05, Wilcoxon rank-sum tests. f, Similar increases in SRP-bound reads were observed for certain non-secretory proteins as exemplified by phosphoacetylglucosamine mutase (PCM1) and tRNASer Um44 2′-O-methyltransferase (TRM44). Hydrophobic sequences in non-secretory proteins, coupled with attenuation of elongation, may lead to SRP recruitment.

Mentions: While elongation arrest is not a general consequence of SRP binding in vivo, recent work showed that a rare-codon directed slowdown of elongation facilitates SRP binding19. An intrinsic, non-SRP-dependent elongation slowdown should increase ribosome-protected reads at the same codon in both soluble SRP-bound polysomes and membrane-bound polysomes. Indeed, several transcripts presented such local increases in ribosome-protected reads at sites corresponding to exposure of a targeting signal on the ribosome (Extended Data Fig. 3a–c). Distinct elongation attenuation mechanisms observed at these sites included clusters of rare codons19 and stalling polypeptide elements, such as stretches of positively charged amino acids, or proline motifs, positioned within the exit tunnel20,21. While most secretory transcripts were not significantly enriched in these attenuator elements compared to the proteome (Extended Data Fig. 3d, e), the few non-secretory proteins that cotranslationally bound to SRP were enriched in elongation attenuation elements positioned at sites that exposed a near-cognate hydrophobic sequence for SRP-binding (Extended Data Fig. 3d, f). We speculate the presence of such elements enhances SRP recognition of the near-cognate hydrophobic tracts in these non-secretory proteins.


Cotranslational signal independent SRP preloading during membrane targeting
Elongation pausing and local SRP recruitmenta, b, Local increases of ribosome-protected reads from membrane-bound polysomes, indicated with orange lines, were coincident with rare codons, as in cell division cycle protein 1 (CDC1, a) or polybasic nascent chains, as in the plasma membrane G-protein coupled receptor (GPR1, b). Soluble SRP-bound polysome-protected reads were further increased at the same positions. c, In these cases, hydrophobic sequences in the nascent chain were exposed to the cytosol at the locations of increased reads, which were coincident with elongation attenuators. d, Translational efficiencies for the 6 codons following, and the number of stalling residues within the 10 residues preceding, the sites of increased SRP-bound ribosome reads. Translational efficiency was determined by attributing the nTE score to each codon55. Residues that were found to stall the ribosome, based on previous investigation20,56,57, were lysine, arginine, glutamate, aspartate, proline and glycine. Because of variation in specific motifs, and uncertainty in whether these motifs are additive, we simply compared the total number of these residues in the indicated 10 residue spans. Sets of 10,000 random sequences, at least 10 amino acids from the stop codon, were sampled from 5907 non-dubious ORFs, and translational efficiency and stalling residues were determined over 6 or 10 codon spans. *p ≤ 0.05, **p ≤ 0.01, Wilcoxon rank-sum tests. e, The targeting signals that recruited SRP directly to the nascent chain unusually far from the encoding of the signal had SRP-binding sites coincident with intrinsic elongation attenuation. Secretory protein transcripts that showed an increase in SRP-bound protected reads (see Extended Data Fig. 2c, f) were further classified by the position of the peak relative to the first signal codon. Transcripts with peaks found at least 80 codons after the signal had significantly lower translational efficiency in the 6 codons following the peak. These transcripts also had a greater, but not statistically significant, amount of stalling amino acids in the 10 residues preceding the peak. *p ≤ 0.05, Wilcoxon rank-sum tests. f, Similar increases in SRP-bound reads were observed for certain non-secretory proteins as exemplified by phosphoacetylglucosamine mutase (PCM1) and tRNASer Um44 2′-O-methyltransferase (TRM44). Hydrophobic sequences in non-secretory proteins, coupled with attenuation of elongation, may lead to SRP recruitment.
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Related In: Results  -  Collection

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Figure 7: Elongation pausing and local SRP recruitmenta, b, Local increases of ribosome-protected reads from membrane-bound polysomes, indicated with orange lines, were coincident with rare codons, as in cell division cycle protein 1 (CDC1, a) or polybasic nascent chains, as in the plasma membrane G-protein coupled receptor (GPR1, b). Soluble SRP-bound polysome-protected reads were further increased at the same positions. c, In these cases, hydrophobic sequences in the nascent chain were exposed to the cytosol at the locations of increased reads, which were coincident with elongation attenuators. d, Translational efficiencies for the 6 codons following, and the number of stalling residues within the 10 residues preceding, the sites of increased SRP-bound ribosome reads. Translational efficiency was determined by attributing the nTE score to each codon55. Residues that were found to stall the ribosome, based on previous investigation20,56,57, were lysine, arginine, glutamate, aspartate, proline and glycine. Because of variation in specific motifs, and uncertainty in whether these motifs are additive, we simply compared the total number of these residues in the indicated 10 residue spans. Sets of 10,000 random sequences, at least 10 amino acids from the stop codon, were sampled from 5907 non-dubious ORFs, and translational efficiency and stalling residues were determined over 6 or 10 codon spans. *p ≤ 0.05, **p ≤ 0.01, Wilcoxon rank-sum tests. e, The targeting signals that recruited SRP directly to the nascent chain unusually far from the encoding of the signal had SRP-binding sites coincident with intrinsic elongation attenuation. Secretory protein transcripts that showed an increase in SRP-bound protected reads (see Extended Data Fig. 2c, f) were further classified by the position of the peak relative to the first signal codon. Transcripts with peaks found at least 80 codons after the signal had significantly lower translational efficiency in the 6 codons following the peak. These transcripts also had a greater, but not statistically significant, amount of stalling amino acids in the 10 residues preceding the peak. *p ≤ 0.05, Wilcoxon rank-sum tests. f, Similar increases in SRP-bound reads were observed for certain non-secretory proteins as exemplified by phosphoacetylglucosamine mutase (PCM1) and tRNASer Um44 2′-O-methyltransferase (TRM44). Hydrophobic sequences in non-secretory proteins, coupled with attenuation of elongation, may lead to SRP recruitment.
Mentions: While elongation arrest is not a general consequence of SRP binding in vivo, recent work showed that a rare-codon directed slowdown of elongation facilitates SRP binding19. An intrinsic, non-SRP-dependent elongation slowdown should increase ribosome-protected reads at the same codon in both soluble SRP-bound polysomes and membrane-bound polysomes. Indeed, several transcripts presented such local increases in ribosome-protected reads at sites corresponding to exposure of a targeting signal on the ribosome (Extended Data Fig. 3a–c). Distinct elongation attenuation mechanisms observed at these sites included clusters of rare codons19 and stalling polypeptide elements, such as stretches of positively charged amino acids, or proline motifs, positioned within the exit tunnel20,21. While most secretory transcripts were not significantly enriched in these attenuator elements compared to the proteome (Extended Data Fig. 3d, e), the few non-secretory proteins that cotranslationally bound to SRP were enriched in elongation attenuation elements positioned at sites that exposed a near-cognate hydrophobic sequence for SRP-binding (Extended Data Fig. 3d, f). We speculate the presence of such elements enhances SRP recognition of the near-cognate hydrophobic tracts in these non-secretory proteins.

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