A Genetic Screen for Pre-mRNA Splicing Mutants of Arabidopsis thaliana Identifies Putative U1 snRNP Components RBM25 and PRP39a

Abstract

In a genetic screen for mutants showing modified splicing of an alternatively spliced GFP reporter gene in Arabidopsis thaliana, we identified mutations in genes encoding the putative U1 small nuclear ribonucleoprotein (snRNP) factors RBM25 and PRP39a. The latter has not yet been studied for its role in pre-messenger RNA (pre-mRNA) splicing in plants. Both proteins contain predicted RNA-binding domains and have been implicated in 5′ splice site selection in yeast and metazoan cells. In rbm25 mutants, splicing efficiency of GFP pre-mRNA was reduced and GFP protein levels lowered relative to wild-type plants. By contrast, prp39a mutants exhibited preferential splicing of a U2-type AT-AC intron in GFP pre-mRNA and elevated levels of GFP protein. These opposing findings indicate that impaired function of either RBM25 or PRP39a can differentially affect the same pre-mRNA substrate. Given a prior genome-wide analysis of alternative splicing in rbm25 mutants, we focused on examining the alternative splicing landscape in prp39a mutants. RNA-seq experiments performed using two independent prp39a alleles revealed hundreds of common genes undergoing changes in alternative splicing, including PRP39a itself, a second putative U1 snRNP component PRP40b, and genes encoding a number of general transcription-related proteins. The prp39a mutants displayed somewhat delayed flowering, shorter stature, and reduced seed set but no other obvious common defects under normal conditions. Mutations in PRP39b, the paralog of PRP39a, did not visibly alter GFP expression, indicating the paralogs are not functionally equivalent in this system. Our study provides new information on the contribution of PRP39a to alternative splicing and expands knowledge of plant splicing factors.

The U1 snRNP Subunit LUC7 Modulates Plant Development and Stress Responses via Regulation of Alternative Splicing

“…PRP39, PRP40, and LETHAL UNLESS CBC7 (LUC7) are encoded by small gene families, which will require the generation of multiple mutants for functional studies. Some U1-specific Arabidopsis mutants have been characterized: Mutations in the accessory factor PRP39A cause delayed flowering, probably due to increased expression of the flowering time regulator FLOWERING LOCUS C, but the mutants do not exhibit severe development defects (Wang et al, 2007;Kanno et al, 2017). Nonetheless, prp39a mutants accumulate misspliced mRNAs containing mainly retained introns or skipped exons (Kanno et al, 2017).…”

Heterogeneous Nuclear Ribonucleoprotein H1 Coordinates with Phytochrome and the U1 snRNP Complex to Regulate Alternative Splicing in Physcomitrella patens

“…U1-specific proteins, such as PRP39, are critical for the formation and stability of the U1 snRNP-pre-mRNA complex (Lockhart and Rymond, 1994;McLean and Rymond, 1998;Zhang and Rosbash, 1999). The Arabidopsis prp39 mutant exhibits preferential splicing of the U2-type AT-AC intron (Wang et al, 2007;Kanno et al, 2017), suggesting that plant PRP39 might also function in splice site recognition by U1 snRNP. U1C stabilizes the binding between U1 snRNP and the 59ss (Heinrichs et al, 1990).…”

“…More than 95% of the reads were perfectly aligned to the reference genome. The three major types of AS events, IR, AltD/A, and ES, were analyzed as described previously with minor modifications (http://rackj.sourceforge.net; Kanno et al, 2017). In brief, IR was measured by comparing IR levels in the dark controls (biological repeats from three independent experiments) with those in the R1 replicates using Student's t test, where the IR level was defined as the average read coverage depth of the intron divided by the average read coverage depth of the neighboring exons.…”

“…For example, PRP39, which was first identified in yeast (Saccharomyces cerevisiae), is required for the formation of a U1 snRNP complex as well as the splicing process (Lockhart and Rymond, 1994). In plants, homologs of PRP39 affect flowering time in Arabidopsis and the orchid Doritaenopsis (Wang et al, 2007;Sun et al, 2012;Kanno et al, 2017). Although PRP39 is conserved among species, whether it is involved in regulating pre-mRNA splicing in plants remains unclear.…”

The U1 snRNP subunit LUC7 controls plant development and stress response through alternative splicing regulation

“…On the other hand, proteins such as PRP39, PRP40 94 and LUC7 are encoded by small gene families, which require the generation of 95 multiple mutants for functional studies. Some U1 specific Arabidopsis mutants have 96 been characterized: Mutations in the accessory factor PRP39A cause delayed 97 flowering due to increased expression of the flowering time regulator FLOWERING 98 LOCUS C (FLC), but the mutants do not exhibit severe developmental defects (Wang 99 et al, 2007;Kanno et al, 2017). In a reverse genetic approach, U1-70K expression 100 was specifically reduced in flowers by an antisense RNA and the resulting transgenic Here, we report on the functional characterization of Arabidopsis mutants 109 impaired in U1 snRNP function.…”