snRNA 3′ End Processing by a CPSF73-Containing Complex Essential for Development in Arabidopsis

Abstract

Uridine-rich small nuclear RNAs (snRNAs) are the basal components of the spliceosome and play essential roles in splicing. The biogenesis of the majority of snRNAs involves 3′ end endonucleolytic cleavage of the nascent transcript from the elongating DNA-dependent RNA ploymerase II. However, the protein factors responsible for this process remain elusive in plants. Here, we show that DEFECTIVE in snRNA PROCESSING 1 (DSP1) is an essential protein for snRNA 3′ end maturation in Arabidopsis. A hypomorphic dsp1-1 mutation causes pleiotropic developmental defects, impairs the 3′ end processing of snRNAs, increases the levels of snRNA primary transcripts (pre-snRNAs), and alters the occupancy of Pol II at snRNA loci. In addition, DSP1 binds snRNA loci and interacts with Pol-II in a DNA/RNA-dependent manner. We further show that DSP1 forms a conserved complex, which contains at least four additional proteins, to catalyze snRNA 3′ end maturation in Arabidopsis. The catalytic component of this complex is likely the cleavage and polyadenylation specificity factor 73 kDa-I (CSPF73-I), which is the nuclease cleaving the pre-mRNA 3′ end. However, the DSP1 complex does not affect pre-mRNA 3′ end cleavage, suggesting that plants may use different CPSF73-I-containing complexes to process snRNAs and pre-mRNAs. This study identifies a complex responsible for the snRNA 3′ end maturation in plants and uncovers a previously unknown function of CPSF73 in snRNA maturation.

DSP1 and DSP4 Act Synergistically in Small Nuclear RNA 3′ End Maturation and Pollen Growth

“…Neither dsp1-1 nor dsp4-1 altered the abundance of mature snRNAs (Fig. 4, B and E), consistent with observations in other dsp mutants and in int metazoan mutants (Tao et al, 2009;Liu et al, 2016). Interestingly, both RT-qPCR and northern-blot analyses showed that the levels of mature snRNAs were reduced in dsp1-1 dsp4-1 compared with the wild type and single mutants (Fig.…”

“…The ratio of heterozygous versus wild-type plants in the progeny of DSP4/dsp4-1 crosses was 1.24:1, which is less than the 2:1 ratio expected by Mendelian inheritance (Supplemental Table S1). This result suggested that, like DSP1 (Liu et al, 2016), DSP4 may also reduce gametophyte transmission. To determine whether dsp4-1 affects female or male gametophyte transmission, we performed reciprocal crosses between DSP4/ dsp4-1 and the wild type.…”

“…Supporting this, DSP1 was shown to bind snRNA gene promoters. Furthermore, all available null dsp1-2, dsp2-1, dsp3-2, and cpsf73-1 mutants are embryonically lethal, while dsp1 and cpsf73-I have defective pollen development (Xu et al, 2006;Liu et al, 2016). These observations suggest that the DSP1 complex plays multiple important roles in development.…”

“…In plants, the Defective in snRNA Processing 1 (DSP1) complex and the CPSF complex, both of which contain CPSF73-I, are responsible for the 39-end cleavage of pre-snRNAs and pre-mRNAs, respectively. The DSP1 complex is composed of at least four additional subunits, DSP1 to DSP4 (Liu et al, 2016). Disruption of DSP1, DSP3, DSP4, or CPSF73-I, but not DSP2, impairs pre-snRNA processing, resulting in increased accumulation of pre-snRNAs.…”

“…While DSP4 has sequence similarities with INT9, it does not interact with CPSF73-I, a homolog of INT11 (Liu et al, 2016). Until now, no null DSP4 mutant allele has been analyzed, and the function of DSP4 in pre-snRNA 39 maturation and development is still not understood.…”