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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 membrane enrichmenta, Crude lysates were fractionated, and then polysomes were recovered by sucrose gradient ultracentrifugation and used for ribosome profiling. b, Enrichment of ribosome-protected mRNA reads in the membrane polysome fractions over the soluble polysome fractions from two biological replicates. Every dot represents one ORF. c, Metagene plots of soluble polysome ribosome-protected reads of transcripts encoding proteins lacking ER targeting signals (top), or of membrane-bound polysome protected reads of transcripts encoding secretory proteins that were at least 2-fold membrane-enriched (bottom). For each ORF, ribosome-protected reads at each position were scaled by dividing by the mean reads per codon of the ORF, excluding first two and last two sense codons. The median scaled reads at each position are plotted as a line, and the interquartile range is shaded in gray. d, Ribosome-protected reads at each codon of an example secreted protein, β-1,3-glucanosyltransferase (GAS1), a model SRP-independent protein12. Topology is indicated above, with the signal sequence in lavender. The position where the signal begins to emerge from the ribosome exit tunnel is indicated. e, The number of codons remaining after the encoding of the first residue of a SS, and the corresponding membrane enrichment per SS containing ORF. Signal sequences were divided between those that bind Ssh1p directly upon exposure and those that require a looped conformation (>90 codons after the first SS codon) 16. f, Transcripts remain at the membrane by subsequent translocon binding, thus the small soluble fraction comprises mRNA undergoing initial targeting.
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Figure 5: Cotranslational membrane enrichmenta, Crude lysates were fractionated, and then polysomes were recovered by sucrose gradient ultracentrifugation and used for ribosome profiling. b, Enrichment of ribosome-protected mRNA reads in the membrane polysome fractions over the soluble polysome fractions from two biological replicates. Every dot represents one ORF. c, Metagene plots of soluble polysome ribosome-protected reads of transcripts encoding proteins lacking ER targeting signals (top), or of membrane-bound polysome protected reads of transcripts encoding secretory proteins that were at least 2-fold membrane-enriched (bottom). For each ORF, ribosome-protected reads at each position were scaled by dividing by the mean reads per codon of the ORF, excluding first two and last two sense codons. The median scaled reads at each position are plotted as a line, and the interquartile range is shaded in gray. d, Ribosome-protected reads at each codon of an example secreted protein, β-1,3-glucanosyltransferase (GAS1), a model SRP-independent protein12. Topology is indicated above, with the signal sequence in lavender. The position where the signal begins to emerge from the ribosome exit tunnel is indicated. e, The number of codons remaining after the encoding of the first residue of a SS, and the corresponding membrane enrichment per SS containing ORF. Signal sequences were divided between those that bind Ssh1p directly upon exposure and those that require a looped conformation (>90 codons after the first SS codon) 16. f, Transcripts remain at the membrane by subsequent translocon binding, thus the small soluble fraction comprises mRNA undergoing initial targeting.

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, Crude lysates were fractionated, and then polysomes were recovered by sucrose gradient ultracentrifugation and used for ribosome profiling. b, Enrichment of ribosome-protected mRNA reads in the membrane polysome fractions over the soluble polysome fractions from two biological replicates. Every dot represents one ORF. c, Metagene plots of soluble polysome ribosome-protected reads of transcripts encoding proteins lacking ER targeting signals (top), or of membrane-bound polysome protected reads of transcripts encoding secretory proteins that were at least 2-fold membrane-enriched (bottom). For each ORF, ribosome-protected reads at each position were scaled by dividing by the mean reads per codon of the ORF, excluding first two and last two sense codons. The median scaled reads at each position are plotted as a line, and the interquartile range is shaded in gray. d, Ribosome-protected reads at each codon of an example secreted protein, β-1,3-glucanosyltransferase (GAS1), a model SRP-independent protein12. Topology is indicated above, with the signal sequence in lavender. The position where the signal begins to emerge from the ribosome exit tunnel is indicated. e, The number of codons remaining after the encoding of the first residue of a SS, and the corresponding membrane enrichment per SS containing ORF. Signal sequences were divided between those that bind Ssh1p directly upon exposure and those that require a looped conformation (>90 codons after the first SS codon) 16. f, Transcripts remain at the membrane by subsequent translocon binding, thus the small soluble fraction comprises mRNA undergoing initial targeting.
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Figure 5: Cotranslational membrane enrichmenta, Crude lysates were fractionated, and then polysomes were recovered by sucrose gradient ultracentrifugation and used for ribosome profiling. b, Enrichment of ribosome-protected mRNA reads in the membrane polysome fractions over the soluble polysome fractions from two biological replicates. Every dot represents one ORF. c, Metagene plots of soluble polysome ribosome-protected reads of transcripts encoding proteins lacking ER targeting signals (top), or of membrane-bound polysome protected reads of transcripts encoding secretory proteins that were at least 2-fold membrane-enriched (bottom). For each ORF, ribosome-protected reads at each position were scaled by dividing by the mean reads per codon of the ORF, excluding first two and last two sense codons. The median scaled reads at each position are plotted as a line, and the interquartile range is shaded in gray. d, Ribosome-protected reads at each codon of an example secreted protein, β-1,3-glucanosyltransferase (GAS1), a model SRP-independent protein12. Topology is indicated above, with the signal sequence in lavender. The position where the signal begins to emerge from the ribosome exit tunnel is indicated. e, The number of codons remaining after the encoding of the first residue of a SS, and the corresponding membrane enrichment per SS containing ORF. Signal sequences were divided between those that bind Ssh1p directly upon exposure and those that require a looped conformation (>90 codons after the first SS codon) 16. f, Transcripts remain at the membrane by subsequent translocon binding, thus the small soluble fraction comprises mRNA undergoing initial targeting.
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