Translation of pre-spliced RNAs in the nuclear compartment generates peptides for the MHC class I pathway

Abstract

The scanning of maturing mRNAs by ribosomes plays a key role in the mRNA quality control process. When ribosomes first engage with the newly synthesized mRNA, and if peptides are produced, is unclear, however. Here we show that ribosomal scanning of prespliced mRNAs occurs in the nuclear compartment, and that this event produces peptide substrates for the MHC class I pathway. Inserting antigenic peptide sequences in introns that are spliced out before the mRNAs exit the nuclear compartment results in an equal amount of antigenic peptide products as when the peptides are encoded from the main open reading frame (ORF). Taken together with the detection of intron-encoded nascent peptides and RPS6/RPL7-carrying complexes in the perinucleolar compartment, these results show that peptides are produced by a translation event occurring before mRNA splicing. This suggests that ribosomes occupy and scan mRNAs early in the mRNA maturation process, and suggests a physiological role for nuclear mRNA translation, and also helps explain how the immune system tolerates peptides derived from tissue-specific mRNA splice variants.

Keywords: MHC class I restricted antigen presentation; mRNA maturation; nuclear translation.

Fig. 1.

Detection of intron and exon RNA sequences in the nuclear and cytoplasmic compartments. (A) Cartoon illustrating the different positions of the SL8 antigenic epitope in the exon or introns of the Globin gene constructs. The position of the PTC that triggers NMD in the Glob-exon-SL8-PTC and the Glob-intron1-proximal-SL8-PTC constructs and primer locations used for RT-PCR are indicated. A list of the constructs used is provided in Fig. S1A. (B) RNA FISH assays using a mix of exon and intron probes (Upper Left) showing the Glob-exon-SL8RNA in the nuclear and cytoplasmic compartments. Exon probes alone (Upper Right and Lower Right) detected the presence of exon RNA sequences in the cytoplasm and nucleus. The introduction of a PTC resulted in loss of exon staining in the cytoplasm (Lower Right). Intron-specific probes (Lower Left) detected RNA in the nuclear compartment only (Fig. S2). (C) Subcellular fractionation followed by RT-PCR using exon-junction primers showing that the introduction of a PTC codon triggers NMD and depletes mRNA levels in both the nuclear and cytoplasmic compartments (Fig. S3A). The graph shows the average of three experiments with Glob-exon-SL8 mRNA levels set to 100%. (D) Western blot showing that the presence of the PTC prevents canonical mRNA translation and the synthesis of full-length proteins.

Fig. 2.

Antigenic peptides for the MHC class I pathway are produced from nonspliced mRNAs. (A) Increasing amounts of DNAs expressing indicated Globin gene constructs in HEK293^Kb cells followed by incubation with the SL8-specific CD8⁺ T-cell hybridoma (B3Z) revealed the production of the SL8 epitope derived from PTPs irrespective of whether the epitope was introduced in the intron or exon sequence or whether the mRNAs carry a PTC. (B) Introducing the SL8 epitope in either the distal region of intron 1 or intron 2 led to an ∼40% reduction in antigen presentation compared with that in the proximal region of intron 1 or in exon 1. (C) Changing the SL8 epitope for the MBP epitope presented on K^k MHC class I molecules and detected by specific CD8⁺ T cells showed a similar profile as the SL8 epitope. (D) A single nucleotide frame shift inserted before the SL8 epitope resulted in an ∼100-fold reduction in antigen presentation. The data show the average of at least three independent experiments with SD minus the values from mock-transfected cells. Free SL8 peptide was added to cells to ensure that T-cell assays were carried out at nonsaturated conditions and that the expression of MHC class I molecules was not affected.

Fig. 3.

Preventing mRNA nuclear export increases the production of MHC class I substrates. (A) Fusing the RRE sequence of HIV-1 to the 3′ UTR of the Globin gene targeted nonspliced mRNAs for nuclear export via the CRM1 pathway in a Rev-dependent fashion, allowing the detection of intron sequences in the cytoplasm using RNA FISH (Left and Center) or by RT-PCR from nuclear and cytoplasmic cellular fractions (Right). NT, nontransfected control cells. (B) Increasing levels of Rev resulted in a subsequent decrease in the relative amount of Glob-exon-SL8-RRE mRNA in the nucleus and a concomitant decrease in the amount of antigenic peptides produced. (C) CRM1-mediated export is blocked by the LMB compound and treating cells with increasing concentrations of LMB prevents Rev-mediated RNA export, with a subsequent increase in the relative amount of RNA in the nucleus and an increase in antigen presentation. (D) The Rev protein does not bind the IFN-α1 mRNA, but interferes with its nuclear export via CRM1. Increasing amounts of Rev led to accumulation of the IFN-α1-SL8 mRNA in the nucleus and to an increase in antigen presentation. (E) Treatment of cells with the spliceosome inhibitor isoginkgetin resulted in an increase in antigen presentation when the SL8 epitope was inserted in Globin gene constructs, but not in the context of cDNA (Fig. S3D).

Fig. 4.

PTPs are produced in the nuclear compartment. (A) The antigenic peptide epitope was exchanged for the HA epitope in indicated β-Globin gene constructs and used to visualize PTPs by immunohistochemistry analysis. Treatment of cells with the proteasome inhibitor epoxomycin for 15 or 30 min increased the amount of HA epitopes detected in the cytoplasmic compartment. (B) The compound puromycin is incorporated in the nascent peptide and, together with the elongation inhibitor emetine, can be used to visualize nascent peptides attached to the ribosome using anti-puromycin antibodies (27). Expression of the HA epitope from within an intron (Glob-intron-HA-PTC), followed by fixation and anti-puromycin (mouse mAb) and anti-HA (rabbit polyclonal sera) shows colocalization in the nucleus but not in the cytoplasm. (C) The PLA allows identification of two primary antibodies in close proximity. Secondary PLA-labeled antibodies revealed a specific signal toward anti-puromycin and anti-HA antibodies in the nuclear and cytoplasmic compartments from the Glob-exon-HA construct (Upper) and in the nuclear compartment using Glob-intron-HA-PTC (Lower). (D) (Left) Immunohistochemistry detected S6 of the small ribosomal subunit in the cytoplasm, nucleoli, and perinucleoli. (Center) The combination of anti-HA and anti-S6 showed a nuclear PLA reaction only. (Right) Anti-S6 (mouse Ab) and anti-L7 of the large subunit (polyclonal Ab) showed PLA complexes in the cytoplasmic and nuclear compartments. The S6 and L7 PLA complexes were not detected in nucleoli despite the large amount of respective proteins in this compartment (Fig. S4D).