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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.


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Ribosome profiling of SRP-bound monosomesa, Ribosome-protected reads, in tags per million (TPM) for each ORF, from SRP-bound monosome fractions from two biological replicates. b, Ribosome-protected reads from the soluble SRP-bound monosome and SRP-bound polysome fractions of the same biological replicate, with cycloheximide treatment. c, Distribution of ribosome reads within example ORFs that display SRP-bound monosome and polysome profiles consistent with direct recognition of the nascent chain. d, If RNCs can recruit SRP while a TMD is within the exit tunnel, then there will be an increase in ribosome-protected reads from SRP-bound monosomes when the TMD begins to translate (lavender). This increase will maximize when the TMD is exposed to the cytosol (orange). e, Distribution of ribosome reads within example ORFs that display SRP-bound monosome profiles consistent with recruitment to transcripts prior to targeting signal synthesis. Examples are arranged for an increasing distance from the start codon to the first TMD. Only the first 600 codons for each ORF are shown.
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Figure 9: Ribosome profiling of SRP-bound monosomesa, Ribosome-protected reads, in tags per million (TPM) for each ORF, from SRP-bound monosome fractions from two biological replicates. b, Ribosome-protected reads from the soluble SRP-bound monosome and SRP-bound polysome fractions of the same biological replicate, with cycloheximide treatment. c, Distribution of ribosome reads within example ORFs that display SRP-bound monosome and polysome profiles consistent with direct recognition of the nascent chain. d, If RNCs can recruit SRP while a TMD is within the exit tunnel, then there will be an increase in ribosome-protected reads from SRP-bound monosomes when the TMD begins to translate (lavender). This increase will maximize when the TMD is exposed to the cytosol (orange). e, Distribution of ribosome reads within example ORFs that display SRP-bound monosome profiles consistent with recruitment to transcripts prior to targeting signal synthesis. Examples are arranged for an increasing distance from the start codon to the first TMD. Only the first 600 codons for each ORF are shown.

Mentions: To understand the basis for the specificity of SRP in vivo, we next determined the initial point of SRP recruitment to ribosomes translating secretory proteins. Since polysomes require only a single SRP-bound ribosome to co-purify with Srp72p, additional strategies were necessary to identify mRNA footprints originated from a single SRP-bound ribosome. We developed a protocol using in vivo monosomes to identify the initial SRP binding event on RNCs (Fig. 2a). At any given time, a fraction of transcripts contain only a single actively translating ribosome (Extended Data Fig. 4a). Total soluble monosomes yield a similar distribution of protected reads compared to polysomes (Extended Data Fig. 4b–e and Supplementary Discussion). We separated soluble SRP-bound monosomes from SRP-bound polysomes and subjected both fractions to Ribo-seq analysis (Extended Data Fig. 5a–b). Of note, the monosomes were necessarily bound to SRP during the purification, and thus should reveal which codons are responsible for the initial SRP recruitment step.


Cotranslational signal independent SRP preloading during membrane targeting
Ribosome profiling of SRP-bound monosomesa, Ribosome-protected reads, in tags per million (TPM) for each ORF, from SRP-bound monosome fractions from two biological replicates. b, Ribosome-protected reads from the soluble SRP-bound monosome and SRP-bound polysome fractions of the same biological replicate, with cycloheximide treatment. c, Distribution of ribosome reads within example ORFs that display SRP-bound monosome and polysome profiles consistent with direct recognition of the nascent chain. d, If RNCs can recruit SRP while a TMD is within the exit tunnel, then there will be an increase in ribosome-protected reads from SRP-bound monosomes when the TMD begins to translate (lavender). This increase will maximize when the TMD is exposed to the cytosol (orange). e, Distribution of ribosome reads within example ORFs that display SRP-bound monosome profiles consistent with recruitment to transcripts prior to targeting signal synthesis. Examples are arranged for an increasing distance from the start codon to the first TMD. Only the first 600 codons for each ORF are shown.
© Copyright Policy - permission-link
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5120976&req=5

Figure 9: Ribosome profiling of SRP-bound monosomesa, Ribosome-protected reads, in tags per million (TPM) for each ORF, from SRP-bound monosome fractions from two biological replicates. b, Ribosome-protected reads from the soluble SRP-bound monosome and SRP-bound polysome fractions of the same biological replicate, with cycloheximide treatment. c, Distribution of ribosome reads within example ORFs that display SRP-bound monosome and polysome profiles consistent with direct recognition of the nascent chain. d, If RNCs can recruit SRP while a TMD is within the exit tunnel, then there will be an increase in ribosome-protected reads from SRP-bound monosomes when the TMD begins to translate (lavender). This increase will maximize when the TMD is exposed to the cytosol (orange). e, Distribution of ribosome reads within example ORFs that display SRP-bound monosome profiles consistent with recruitment to transcripts prior to targeting signal synthesis. Examples are arranged for an increasing distance from the start codon to the first TMD. Only the first 600 codons for each ORF are shown.
Mentions: To understand the basis for the specificity of SRP in vivo, we next determined the initial point of SRP recruitment to ribosomes translating secretory proteins. Since polysomes require only a single SRP-bound ribosome to co-purify with Srp72p, additional strategies were necessary to identify mRNA footprints originated from a single SRP-bound ribosome. We developed a protocol using in vivo monosomes to identify the initial SRP binding event on RNCs (Fig. 2a). At any given time, a fraction of transcripts contain only a single actively translating ribosome (Extended Data Fig. 4a). Total soluble monosomes yield a similar distribution of protected reads compared to polysomes (Extended Data Fig. 4b–e and Supplementary Discussion). We separated soluble SRP-bound monosomes from SRP-bound polysomes and subjected both fractions to Ribo-seq analysis (Extended Data Fig. 5a–b). Of note, the monosomes were necessarily bound to SRP during the purification, and thus should reveal which codons are responsible for the initial SRP recruitment step.

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