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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.


The role translation in membrane enrichmenta, Lysates were treated with puromycin prior to membrane fractionation. mRNA recovered from the soluble and membrane fractions were used for RNA-seq b, Membrane enrichment of secretory protein transcripts (SS, TMD, SS-TMD, or TA, n = 729) following puromycin treatment of lysates.
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Figure 11: The role translation in membrane enrichmenta, Lysates were treated with puromycin prior to membrane fractionation. mRNA recovered from the soluble and membrane fractions were used for RNA-seq b, Membrane enrichment of secretory protein transcripts (SS, TMD, SS-TMD, or TA, n = 729) following puromycin treatment of lysates.

Mentions: We next examined if the membrane association of secretory transcripts similarly depends on ongoing translation. In principle, ER-localized proteins could recruit secretory transcripts to the membrane in the absence of translation26,28 (Fig. 4b). Membrane and soluble mRNAs were fractionated in the presence and absence of puromycin treatment and subject to RNA-seq analysis (Extended Data Fig. 7). Disruption of translating ribosomes reduced membrane enrichment for all secretory protein transcripts, including those of PMP1 and PMP2. This result was confirmed using the GFP-PMP1 reporter (Extended Data Fig. 6h).


Cotranslational signal independent SRP preloading during membrane targeting
The role translation in membrane enrichmenta, Lysates were treated with puromycin prior to membrane fractionation. mRNA recovered from the soluble and membrane fractions were used for RNA-seq b, Membrane enrichment of secretory protein transcripts (SS, TMD, SS-TMD, or TA, n = 729) following puromycin treatment of lysates.
© Copyright Policy - permission-link
Related In: Results  -  Collection

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

Figure 11: The role translation in membrane enrichmenta, Lysates were treated with puromycin prior to membrane fractionation. mRNA recovered from the soluble and membrane fractions were used for RNA-seq b, Membrane enrichment of secretory protein transcripts (SS, TMD, SS-TMD, or TA, n = 729) following puromycin treatment of lysates.
Mentions: We next examined if the membrane association of secretory transcripts similarly depends on ongoing translation. In principle, ER-localized proteins could recruit secretory transcripts to the membrane in the absence of translation26,28 (Fig. 4b). Membrane and soluble mRNAs were fractionated in the presence and absence of puromycin treatment and subject to RNA-seq analysis (Extended Data Fig. 7). Disruption of translating ribosomes reduced membrane enrichment for all secretory protein transcripts, including those of PMP1 and PMP2. This result was confirmed using the GFP-PMP1 reporter (Extended Data Fig. 6h).

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.