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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

Translation and the role of SRPa, Distributions of RNA-seq SRP enrichment scores from secretory protein transcripts (SS, TMD, SS-TMD, or TA), with or without puromycin treatment. Included ORFs have at least 2-fold SRP enrichment without puromycin. b, The prt1-1 allele prevents initiation at non-permissive temperatures. Translational run-off removes all ribosomes from transcripts. b, Transcripts are retained on the membrane though binding of the RNC to the translocon. It is also possible that mRNA binding proteins at the ER bind transcripts. c, Distributions of RNA-seq membrane enrichment scores of secretory protein transcripts (n = 584). d, After mRNA export, a pioneer round of targeting directs secretory transcripts to the ER membrane. SRP is specifically pre-recruited transcripts that will present a functional targeting signal. Upon emergence of an SS or TMD, SRP directs RNCs to the ER membrane. Once at the ER membrane, transcripts are retained over multiple rounds of translation.
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Figure 4: Translation and the role of SRPa, Distributions of RNA-seq SRP enrichment scores from secretory protein transcripts (SS, TMD, SS-TMD, or TA), with or without puromycin treatment. Included ORFs have at least 2-fold SRP enrichment without puromycin. b, The prt1-1 allele prevents initiation at non-permissive temperatures. Translational run-off removes all ribosomes from transcripts. b, Transcripts are retained on the membrane though binding of the RNC to the translocon. It is also possible that mRNA binding proteins at the ER bind transcripts. c, Distributions of RNA-seq membrane enrichment scores of secretory protein transcripts (n = 584). d, After mRNA export, a pioneer round of targeting directs secretory transcripts to the ER membrane. SRP is specifically pre-recruited transcripts that will present a functional targeting signal. Upon emergence of an SS or TMD, SRP directs RNCs to the ER membrane. Once at the ER membrane, transcripts are retained over multiple rounds of translation.

Mentions: We assessed the global ribosome dependency of SRP binding to secretory transcripts using either puromycin or CHX incubations to respectively disrupt or stabilize elongating ribosomes. Srp72p-bound transcripts isolated from the soluble fraction were examined by RNA-seq (Fig. 4a). SRP association with all secretory mRNAs was sensitive to puromycin. Transcripts that only recruit SRP through a canonical nascent chain interaction were more dependent on elongating ribosomes. The reduced puromycin sensitivity observed for pre-enriched transcripts may arise from the inability of puromycin to disrupt initiating ribosomes27, which appear able to recruit SRP (Extended Data Fig. 6g).


Cotranslational signal independent SRP preloading during membrane targeting
Translation and the role of SRPa, Distributions of RNA-seq SRP enrichment scores from secretory protein transcripts (SS, TMD, SS-TMD, or TA), with or without puromycin treatment. Included ORFs have at least 2-fold SRP enrichment without puromycin. b, The prt1-1 allele prevents initiation at non-permissive temperatures. Translational run-off removes all ribosomes from transcripts. b, Transcripts are retained on the membrane though binding of the RNC to the translocon. It is also possible that mRNA binding proteins at the ER bind transcripts. c, Distributions of RNA-seq membrane enrichment scores of secretory protein transcripts (n = 584). d, After mRNA export, a pioneer round of targeting directs secretory transcripts to the ER membrane. SRP is specifically pre-recruited transcripts that will present a functional targeting signal. Upon emergence of an SS or TMD, SRP directs RNCs to the ER membrane. Once at the ER membrane, transcripts are retained over multiple rounds of translation.
© Copyright Policy - permission-link
Related In: Results  -  Collection

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

Figure 4: Translation and the role of SRPa, Distributions of RNA-seq SRP enrichment scores from secretory protein transcripts (SS, TMD, SS-TMD, or TA), with or without puromycin treatment. Included ORFs have at least 2-fold SRP enrichment without puromycin. b, The prt1-1 allele prevents initiation at non-permissive temperatures. Translational run-off removes all ribosomes from transcripts. b, Transcripts are retained on the membrane though binding of the RNC to the translocon. It is also possible that mRNA binding proteins at the ER bind transcripts. c, Distributions of RNA-seq membrane enrichment scores of secretory protein transcripts (n = 584). d, After mRNA export, a pioneer round of targeting directs secretory transcripts to the ER membrane. SRP is specifically pre-recruited transcripts that will present a functional targeting signal. Upon emergence of an SS or TMD, SRP directs RNCs to the ER membrane. Once at the ER membrane, transcripts are retained over multiple rounds of translation.
Mentions: We assessed the global ribosome dependency of SRP binding to secretory transcripts using either puromycin or CHX incubations to respectively disrupt or stabilize elongating ribosomes. Srp72p-bound transcripts isolated from the soluble fraction were examined by RNA-seq (Fig. 4a). SRP association with all secretory mRNAs was sensitive to puromycin. Transcripts that only recruit SRP through a canonical nascent chain interaction were more dependent on elongating ribosomes. The reduced puromycin sensitivity observed for pre-enriched transcripts may arise from the inability of puromycin to disrupt initiating ribosomes27, which appear able to recruit SRP (Extended Data Fig. 6g).

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