An ortholog of the Vasa intronic gene is required for small RNA-mediated translation repression in Chlamydomonas reinhardtii

Abstract

Small RNAs (sRNAs) associate with Argonaute (AGO) proteins in effector complexes, termed RNA-induced silencing complexes (RISCs), which regulate complementary transcripts by translation inhibition and/or RNA degradation. In the unicellular alga Chlamydomonas, several metazoans, and land plants, emerging evidence indicates that polyribosome-associated transcripts can be translationally repressed by RISCs without substantial messenger RNA (mRNA) destabilization. However, the mechanism of translation inhibition in a polyribosomal context is not understood. Here we show that Chlamydomonas VIG1, an ortholog of the Drosophila melanogaster Vasa intronic gene (VIG), is required for this process. VIG1 localizes predominantly in the cytosol and comigrates with monoribosomes and polyribosomes by sucrose density gradient sedimentation. A VIG1-deleted mutant shows hypersensitivity to the translation elongation inhibitor cycloheximide, suggesting that VIG1 may have a nonessential role in ribosome function/structure. Additionally, FLAG-tagged VIG1 copurifies with AGO3 and Dicer-like 3 (DCL3), consistent with it also being a component of the RISC. Indeed, VIG1 is necessary for the repression of sRNA-targeted transcripts at the translational level but is dispensable for cleavage-mediated RNA interference and for the association of the AGO3 effector with polyribosomes or target transcripts. Our results suggest that VIG1 is an ancillary ribosomal component and plays a role in sRNA-mediated translation repression of polyribosomal transcripts.

Keywords: RNA interference; RNA silencing; SERBP1; VIG1; microRNA.

Fig. 1.

Chlamydomonas VIG1 is required for the siRNA-mediated translation repression of the MAA7 transcript. (A) Growth and survival of the indicated strains on TAP medium with or without 7 μM 5-fluoroindole. CC-124, wild-type strain; Maa7-IR44, CC-124 transformed with an IR transgene designed to induce RNAi of MAA7; vig1, VIG1 deletion mutant; vig1(tagVIG1)-3 and -6, transgenic strains of vig1 transformed with FLAG-CBP-VIG1 under the control of the PsaD promoter. (B) Immunoblot analysis of tryptophan synthase β-subunit levels. Immunodetection of histone H3 was used as a control for equivalent loading of the lanes. (C) Northern blot analysis of MAA7 transcript levels. The same filter was reprobed with the coding sequence of ACT1 (encoding actin) as a control for similar loading of the lanes. (D) Northern blot analysis of MAA7 siRNAs in the indicated strains. The same filter was reprobed with the U6 small nuclear RNA sequence to assess the amount of sample loaded per lane. (E) Northern blot analyses of sRNAs isolated from the indicated strains and detected with probes specific for Chlamydomonas miRNAs. (F) Immunoblot analysis of AGO3/2 proteins in the indicated strains. The asterisk indicates a nonspecific cross-reacting antigen.

Fig. 2.

Chlamydomonas vig1 mutant is defective in the translation repression, by an endogenous miRNA, of Cre16.g683650 (encoding a predicted protein kinase of unknown function). (A) Ribosome profiling of the Cre16.g683650 transcript. The diagram (Top) depicts the hybridization of miR_C to its recognition site, overlapping the stop codon, in the Cre16.g683650 mRNA. The mismatch to nucleotide 10 of the miRNA would prevent AGO-mediated cleavage of the target transcript. Histograms of the 5′-end positions of ribosome-protected fragments (Ribo-seq; blue) or of normalized total RNA reads (RNA-seq; gray) are shown along the length of the mRNA schematic (CDS is indicated by the orange box). Ribosome-protected fragments were predominantly 27 (or 28) nt in length and their 5′ ends mapped to the second (or first) nucleotide position of codons, consistent with previous genome-wide Ribo-seq analyses in Chlamydomonas (56). The distribution on the transcript of RNA-seq reads serves as a control for a possible technical bias in the detection of certain mRNA sequences. The y axes of the histogram graphs indicate read frequency. (B) Immunoblot and semiquantitative RT-PCR analyses of Cre16.g683650 protein and transcript levels in the indicated strains. Immunodetection of histone H3 was used as a control for equivalent loading of proteins in the lanes. Amplification of ACT1 (encoding actin) transcripts is shown as an RT-PCR input control. CC-124, wild-type strain; Maa7-IR44, CC-124 transformed with an IR transgene designed to induce RNAi of MAA7; vig1, VIG1 deletion mutant; vig1(tagVIG1)-3, transgenic strain of vig1 transformed with FLAG-CBP-VIG1 under the control of the PsaD promoter.

Fig. 3.

VIG1 is a bona fide (mi)RISC component. (A) FLAG-CBP-VIG1–associated proteins, isolated by affinity purification, were separated by SDS/PAGE and visualized by Sypro Ruby staining. The indicated proteins were identified by mass spectrometry analyses. A mock purification with FLAG-CBP–tagged Ble (conferring resistance to bleomycin) is also shown. The asterisk indicates the FLAG-CBP-VIG1 protein. (B) In vitro RISC cleavage assay. The diagram (Top) depicts the homologous RNA substrate, hybridizing to 2 fully complementary MAA7 siRNAs, and the predicted cleavage sites generating ³²P-labeled 5′-RNA fragments of the indicated lengths. FLAG-CBP-VIG1 and associated proteins were affinity purified and incubated with the homologous RNA or with a nonhomologous RNA (non-H RNA) under the denoted conditions. Reaction products were separated on a denaturing polyacrylamide/urea gel and analyzed by autoradiography (Bottom). The homologous RNA substrate was also incubated with purified FLAG-CBP-Ble as a negative control.

Fig. 4.

Immunofluorescence localization of FLAG-CBP–tagged VIG1. Phase-contrast images of the cells, immunolocalization of epitope-tagged VIG1 (detected with an antibody conjugated to Alexa Fluor 488), DAPI staining of nuclear and organellar DNA, and merged images. Representative images are shown, with the location of the nucleus indicated by “N.” Cells were grown to middle logarithmic phase in TAP medium, collected by centrifugation, and then processed for immunofluorescence microscopy. Aliquots of cells were also exposed to 42 °C for 45 min (heat shock) or incubated in the presence of 50 μg/mL cycloheximide for 2 h prior to preparation for immunofluorescence microscopy. vig1(tagVIG1)-3, VIG1 deletion mutant expressing a transgene of FLAG-CBP-VIG1 under the control of the PsaD promoter. Maa7-IR44, parental strain. (Scale bars, 5 μm.)

Fig. 5.

VIG1 is not required for AGO3 association with polyribosomes or target transcripts. (A) Polyribosome profile of a vig1(tagVIG1)-3 lysate treated with 150 μg/mL cycloheximide and fractionated by sucrose density gradient centrifugation (Top). The distribution of the AGO3/2, RPL37, and FLAG-CBP-VIG1 proteins in the gradient fractions was examined by immunoblotting (Bottom). The location of monoribosomes (M) and polyribosomes (Poly) in the profile is shown above the blots. The asterisk indicates a nonspecific cross-reacting antigen. (B) Polyribosome profiles of the Maa7-IR44 and vig1 strains and immunological detection of AGO3/2 and RPL37 distribution in the gradient fractions. (C) RNA-binding protein immunoprecipitation and subsequent RT-PCR detection of AGO3-associated transcripts. RIP with anti–FLAG-M2 agarose beads (FLAG-IP) was performed from lysates of the indicated strains. Input RNA corresponded to 5% of the total purified amount. ACT1 was examined as a control transcript, not targeted by an sRNA-mediated mechanism. Maa7-IR44(FLAG-AGO3)-56, transgenic strain of Maa7-IR44 transformed with FLAG-tagged AGO3; vig1(FLAG-AGO3)-31, transgenic strain of vig1 transformed with FLAG-tagged AGO3; Maa7-IR44(FLAG-Ble), transgenic strain of Maa7-IR44 transformed with FLAG-tagged Ble.