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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 monosomesa, Ribosomes transition from monosomes to polysomes during elongation. The pioneer round of initiation will be a monosome, and during elongation there is a chance of additional initiation converting the transcript to a polysome. Similarly, a polysome will become a monosome if all ribosomes but one terminate. As mRNA is sampled closer to the stop codon, the likelihood of observing a footprint from the final ribosome will increase. b, Metagene analysis of soluble monosome or polysome protected reads from proteins lacking an ER targeting signal. Data were obtained using cycloheximide treatment. ORFs are at least 400 codons long and have an average of at least 0.5 reads per codon in each dataset. For each ORF, ribosome reads at each position were divided by the mean reads per codon over the range +160 to +240 codons. The median normalized read value at each codon position is plotted, and the interquartile range is shaded in gray. c, Relative reads at the start codon from ORFs normalized in b. d, Distributions of the ratio of ribosome-protected reads found in soluble monosomes over soluble polysomes. e, A pioneer round of translation deposits mRNA on the membrane. Polysomes will be retained at the membrane and are therefore depleted from the soluble fraction.
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Figure 8: Ribosome profiling of monosomesa, Ribosomes transition from monosomes to polysomes during elongation. The pioneer round of initiation will be a monosome, and during elongation there is a chance of additional initiation converting the transcript to a polysome. Similarly, a polysome will become a monosome if all ribosomes but one terminate. As mRNA is sampled closer to the stop codon, the likelihood of observing a footprint from the final ribosome will increase. b, Metagene analysis of soluble monosome or polysome protected reads from proteins lacking an ER targeting signal. Data were obtained using cycloheximide treatment. ORFs are at least 400 codons long and have an average of at least 0.5 reads per codon in each dataset. For each ORF, ribosome reads at each position were divided by the mean reads per codon over the range +160 to +240 codons. The median normalized read value at each codon position is plotted, and the interquartile range is shaded in gray. c, Relative reads at the start codon from ORFs normalized in b. d, Distributions of the ratio of ribosome-protected reads found in soluble monosomes over soluble polysomes. e, A pioneer round of translation deposits mRNA on the membrane. Polysomes will be retained at the membrane and are therefore depleted from the soluble fraction.

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 monosomesa, Ribosomes transition from monosomes to polysomes during elongation. The pioneer round of initiation will be a monosome, and during elongation there is a chance of additional initiation converting the transcript to a polysome. Similarly, a polysome will become a monosome if all ribosomes but one terminate. As mRNA is sampled closer to the stop codon, the likelihood of observing a footprint from the final ribosome will increase. b, Metagene analysis of soluble monosome or polysome protected reads from proteins lacking an ER targeting signal. Data were obtained using cycloheximide treatment. ORFs are at least 400 codons long and have an average of at least 0.5 reads per codon in each dataset. For each ORF, ribosome reads at each position were divided by the mean reads per codon over the range +160 to +240 codons. The median normalized read value at each codon position is plotted, and the interquartile range is shaded in gray. c, Relative reads at the start codon from ORFs normalized in b. d, Distributions of the ratio of ribosome-protected reads found in soluble monosomes over soluble polysomes. e, A pioneer round of translation deposits mRNA on the membrane. Polysomes will be retained at the membrane and are therefore depleted from the soluble fraction.
© Copyright Policy - permission-link
Related In: Results  -  Collection

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Figure 8: Ribosome profiling of monosomesa, Ribosomes transition from monosomes to polysomes during elongation. The pioneer round of initiation will be a monosome, and during elongation there is a chance of additional initiation converting the transcript to a polysome. Similarly, a polysome will become a monosome if all ribosomes but one terminate. As mRNA is sampled closer to the stop codon, the likelihood of observing a footprint from the final ribosome will increase. b, Metagene analysis of soluble monosome or polysome protected reads from proteins lacking an ER targeting signal. Data were obtained using cycloheximide treatment. ORFs are at least 400 codons long and have an average of at least 0.5 reads per codon in each dataset. For each ORF, ribosome reads at each position were divided by the mean reads per codon over the range +160 to +240 codons. The median normalized read value at each codon position is plotted, and the interquartile range is shaded in gray. c, Relative reads at the start codon from ORFs normalized in b. d, Distributions of the ratio of ribosome-protected reads found in soluble monosomes over soluble polysomes. e, A pioneer round of translation deposits mRNA on the membrane. Polysomes will be retained at the membrane and are therefore depleted from the soluble fraction.
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