Dataset Information

The ribosomal P-stalk regulates MHC I antigen presentation

ABSTRACT: MHC I antigen processing and presentation (APP) is a highly regulated process that enables CD8+ T cell immunosurveillance. APP begins with the ribosomal synthesis of a source antigen, yet the role of ribosomes, particularly specialized ribosomes, in antigen presentation is poorly understood. Here, we show that the presence of the “P-stalk” on the ribosome enhances antigen presentation. The addition of the P-stalk to the ribosome is stimulated by cytokines that upregulate APP components, and knockdown of one of the P-stalk proteins (P1) reduces T cell recognition of tumor cells. Mechanistically, we show that P1-containing ribosomes exhibit enhanced translation of MHC class I molecules and accessory APP components. Finally, analysis of patient data reveals that the mRNA expression of the P-stalk proteins positively correlates with CD8+ T cell infiltration, a trend not seen for other ribosomal proteins. In all, we demonstrate that the presence of the P-stalk defines a specialized ribosome population that enhances antigen presentation, something that may be exploited by cancer cells to escape immunosurveillance.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Skin

DISEASE(S): Melanoma

SUBMITTER: Onno Bleijerveld

LAB HEAD: Onno Bleijerveld

PROVIDER: PXD041688 | Pride | 2025-12-01

REPOSITORIES: pride

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			Action	DRS
	20211125_Exploris_Project_532_Anna_Dopler_DiaNN_proteome_1A.raw	Raw
	20211125_Exploris_Project_532_Anna_Dopler_DiaNN_proteome_1B.raw	Raw
	20211125_Exploris_Project_532_Anna_Dopler_DiaNN_proteome_1C.raw	Raw
	20211125_Exploris_Project_532_Anna_Dopler_DiaNN_proteome_2A.raw	Raw
	20211125_Exploris_Project_532_Anna_Dopler_DiaNN_proteome_2B.raw	Raw

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Publications

P-stalk ribosomes act as master regulators of cytokine-mediated processes.

Dopler Anna A Alkan Ferhat F Malka Yuval Y van der Kammen Rob R Hoefakker Kelly K Taranto Daniel D Kocabay Naz N Mimpen Iris I Ramirez Christel C Malzer Elke E Isaeva Olga I OI Kerkhoff Mandy M Gangaev Anastasia A Silva Joana J Ramalho Sofia S Hoekman Liesbeth L Altelaar Maarten M Beijersbergen Roderick R Akkari Leila L Yewdell Jonathan Wilson JW Kvistborg Pia P Faller William James WJ

Cell 20241021 24

Inflammatory cytokines are pivotal to immune responses. Upon cytokine exposure, cells enter an "alert state" that enhances their visibility to the immune system. Here, we identified an alert-state subpopulation of ribosomes defined by the presence of the P-stalk. We show that P-stalk ribosomes (PSRs) are formed in response to cytokines linked to tumor immunity, and this is at least partially mediated by P-stalk phosphorylation. PSRs are involved in the preferential translation of mRNAs vital for ...[more]

PMID: 39437780

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Project description:We use mRNA-seq in combination with polysome profiling to determine translational status for all mRNAs in Drosophila mature oocytes and activated eggs. Puromycin-treated lysates are used as a negative control in polysome profiling experiments. Additionally, we use ribosome footprinting to globally measure translational efficiency of mRNAs in wild type mature oocytes as well as wild type and png mutant activated eggs. Lysates of hand-dissected Drosophila mature oocytes (containing ~540 M-NM-<g of total RNA) were subjected to separation by velocity sedimentation through sucrose gradients. In this way, free mRNAs (present in RNPs fraction) or those comigrating with ribosomal subunits (40S or 60S+80S fractions) or with varying numbers of bound ribosomes (low polysomes (2-4 ribosomes), medium polysomes (5-9 ribosomes), and heavy polysomes (more than 10 ribosomes) can be separated based on their size and collected as sucrose gradient fractions. To compare quantitatively the levels of every mRNA across the polysome gradient fractions, we added 5ng of S. cerevisiae mRNA as an exogenous spike-in to each of the six fractions of interest: RNPs, 40S, 60S+80S, low polysomes, medium polysomes and heavy polysomes. RNA was extraced from these fractions, follwing proteinase K treatment, by hot acid phenol method. In case of unfractionated lysates, RNA was extracted using TRIzol (Invitrogen) according to manufacturerM-bM-^@M-^Ys instructions. mRNA-seq samples were prepared from 1 M-NM-<g of total RNA (in case of sucrose gradient fractions and unfractionated lysates) and subject to Illumina based sequencing. Puromycin-treated lysates of mature oocytes or 0-2h Drosophila activated eggs (containing ~540 M-NM-<g of total RNA) were also subjected to separation by velocity sedimentation through sucrose gradients. Puromycin causes premature termination of elongating ribosomes and thus it can be used to determine whether the mRNAs co-sedimenting with the polysomal peaks (defined here as M-bM-^IM-%5 ribosomes) were actively engaged in translation. As an independent approach to assess translation and obtain information on the position of ribosomes on mRNAs, we employed ribosome footprinting. In addition to analyzing the same samples, as by polysome profiling, we also analyzed png mutant activated eggs by ribosome footprinting. Ribosome footprint profiling measures the number of ribosome-protected fragments (RPFs) derived from the mRNAs of each gene, resulting in a singular value of translational efficiency (TE) for each gene (TE=RPF/RNA).