An hnRNP-like RNA-binding protein affects alternative splicing by in vivo interaction with transcripts in Arabidopsis thaliana

Abstract

Alternative splicing (AS) of pre-mRNAs is an important regulatory mechanism shaping the transcriptome. In plants, only few RNA-binding proteins are known to affect AS. Here, we show that the glycine-rich RNA-binding protein AtGRP7 influences AS in Arabidopsis thaliana. Using a high-resolution RT-PCR-based AS panel, we found significant changes in the ratios of AS isoforms for 59 of 288 analyzed AS events upon ectopic AtGRP7 expression. In particular, AtGRP7 affected the choice of alternative 5' splice sites preferentially. About half of the events are also influenced by the paralog AtGRP8, indicating that AtGRP7 and AtGRP8 share a network of downstream targets. For 10 events, the AS patterns were altered in opposite directions in plants with elevated AtGRP7 level or lacking AtGRP7. Importantly, RNA immunoprecipitation from plant extracts showed that several transcripts are bound by AtGRP7 in vivo and indeed represent direct targets. Furthermore, the effect of AtGRP7 on these AS events was abrogated by mutation of a single arginine that is required for its RNA-binding activity. This indicates that AtGRP7 impacts AS of these transcripts via direct interaction. As several of the AS events are also controlled by other splicing regulators, our data begin to provide insights into an AS network in Arabidopsis.

Figure 1.

Changes in the ratio of AS isoforms in AtGRP7-ox, AtGRP8-ox and atgrp7-1 8i. (A) Venn diagram showing the number of splicing events with significant changes in AtGRP7-ox (Col-2) and AtGRP7-ox (C24) when compared to the Col-2 and C24 wild-types, respectively. (B) Venn diagram showing the number of splicing events with significant changes in AtGRP7-ox (Col-2) and AtGRP8-ox (Col-2). (C) Venn diagram showing the number of splicing events with significant changes in AtGRP7-ox (Col-2 and C24) and atgrp7-1 8i. The numbers represent transcripts with significant changes in the ratio of AS isoforms (>5% and P < 0.05).

Figure 2.

Genes/transcripts with significant changes in the AS patterns in opposite directions in atgrp7-1 8i and AtGRP7-ox plants. (A) At2g3600 (#75) Mitochondrial transcription termination factor, (B) At3g29160 (#343) AKIN11, (C) At4g24740 (#227) AFC2, (D) At4g24740 (#226) AFC2 and (E) At5g59950 (#327) Aly/REF-like protein. On the left side of each panel, the percentage of each splice form ± SD based on three biological replicates is indicated for atgrp7-1 8i, wt, and AtGRP7-ox plants, respectively. For comparison, the data for AtGRP8-ox plants are included. On the right side of each panel, the gene and transcript structures and the AS events are shown schematically. Exons are indicated by open boxes and numbered, UTRs—black rectangles, introns—thin lines, splicing events—diagonal lines and stop signs—PTCs. The sizes of the PCR products from each splice isoform are indicated.

Figure 3.

In vivo interaction of AtGRP7-GFP with candidate target transcripts. RIP was performed on plants expressing the AtGRP7-GFP fusion protein under control of the AtGRP7 promoter including 5′- and 3′-UTR and intron in atgrp7-1 (A) and transgenic plants expressing GFP under control of the AtGRP7 promoter including 5′- and 3′-UTR (B). The levels of transcripts co-precipitated in the GFP-Trap® bead precipitate (IP+), the RFP-Trap® bead mock precipitate (IP−) and in the input fractions, respectively, were determined by qRT-PCR in duplicates for At2g21660 (AtGRP7) which served as positive control, At4g39260 (AtGRP8, #90), At3g12570 (FYD, #288), At2g3600 (mTERF, #75), At1g72320 (APUM23, #12), At3g29160 (AKIN11, #343), At5g05550 [transcription factor (TF), #181], At4g24740 (AFC2, #226 and #227) and At5g59950 (Aly/REF-like RNA binding/export factor, #327). Transcript levels were normalized to PP2A and expressed relative to the input. Means ± SD are presented based on three biological and significance was tested using Student’s t-test (**P < 0.005, *P < 0.05). n.d., not detectable; n.s., not significant. In IP− from GFP plants, the transcripts #181 and #327 were not detectable and thus no statistical test was applied.

Figure 4.

AS events in AtGRP7-ox lines not observed in AtGRP7-R⁴⁹Q-ox plants. Left side: RT-PCR analysis of selected genes/transcripts with total RNA from AtGRP7-ox and AtGRP7-RQ-ox plants in both the Col-2 and C24 backgrounds and their respective wt plants. Arrowheads denote RT-PCR products representing AS events whose presence/absence or abundance differs between the over-expression lines with and without the R⁴⁹Q mutation. Right side: gene and transcript structures and the AS events for AtGRP8 (#90), ADA2A (#136), Aly/Ref (#327), AKIN11 (#343) and AFC2 (#226) are shown schematically (see Figure 2). PP2A served as constitutive control.

Figure 5.

Conceptual model depicting common targets of known splicing regulators in Arabidopsis. The impact of AtGRP7, the cap binding complex (CBC) and At-SR30 on selected splicing events, analyzed by the RT-PCR panel, is displayed (33,34). The numbers of the primer pairs for detection of the AS events are indicated. Regulation of the ratio of splice isoforms in the same direction is indicated by ‘=’; regulation in opposite direction is indicated by a line with two arrowheads. For RNA-binding proteins that are influenced by AtGRP7, the name is indicated and dotted arrows indicate a presumed post-transcriptional regulation of yet unknown targets of these proteins. The negative autoregulation of AtGRP7, PTB1 and At-SR30 is depicted (19,31,57). ‘P’ denotes phosphorylation of SR proteins by the LAMMER kinase AFC2 (68). For clarity, the effects of AtGRP8 and At-RS2Z33 on AS events are omitted (see text for details).