Weili Wang, Xuepiao Pu, Siyu Yang, Yujie Feng, Chan Lin, Mu Li, Xi Li, Huali Li, Chunmei Meng, Qingjun Xie, Bin Yu, and Yunfeng Liu

PNAS August 18, 2020 117 (33) 20325-20333

Significance

snRNAs are conserved noncoding RNAs in eukaryotes. Their biogenesis is spatiotemporally regulated in various biological processes. However, related mechanisms regulating snRNA accumulation are less known. DSP1 is an essential component of the DSP1 complex that catalyzes snRNA maturation. Here, we show that DSP1 is subjected to alternative splicing in the pollens, resulting in two splicing variants, DSP1α and DSP1β. DSP1β does not participate in the 3′ maturation of snRNAs but interacts with CPSF73-I and promotes efficient release of CPSF73-I and Poll II from the 3′ end of the snRNA loci and thereby facilitates snRNA transcription termination, resulting in increased snRNA levels in pollens. Our results uncover a mechanism spatiotemporally regulating snRNA biogenesis in plants.

Abstract

Small nuclear RNAs (snRNAs) are the basal components of the spliceosome and play crucial roles in splicing. Their biogenesis is spatiotemporally regulated. However, related mechanisms are still poorly understood. Defective in snRNA processing (DSP1) is an essential component of the DSP1 complex that catalyzes plant snRNA 3′-end maturation by cotranscriptional endonucleolytic cleavage of the primary snRNA transcripts (presnRNAs). Here, we show that DSP1 is subjected to alternative splicing in pollens and embryos, resulting in two splicing variants, DSP1α and DSP1β. Unlike DSP1α, DSP1β is not required for presnRNA 3′-end cleavage. Rather, it competes with DSP1α for the interaction with CPSF73-I, the catalytic subunit of the DSP1 complex, which promotes efficient release of CPSF73-I and the DNA-dependent RNA polymerease II (Pol II) from the 3′ end of snRNA loci thereby facilitates snRNA transcription termination, resulting in increased snRNA levels in pollens. Taken together, this study uncovers a mechanism that spatially regulates snRNA accumulation.

See https://www.pnas.org/content/117/33/20325

Figure 3:

Both DSP1α and DSP1β are required for snRNA biosynthesis in pollens. (A and B) Accumulation of mature snRNAs detected by qRT-PCR (A) or Northern blot (B). (C) Accumulation of presnRNAs detected by qRT-PCR. PresnRNA and mature snRNA levels were normalized to ACT2 and compared with Col in qRT-PCR. Values are means of three biological replicates. Error bars indicate SD. *P < 0.05, **P < 0.01 (Student’s t test). U6 was used as a loading control for Northern blot. (D) In vitro pre-U2.3-polyG 3′-end processing by nuclear protein extracts. The black arrow indicates the input. The gray arrow indicates the mature snRNAs. The relative processing efficiency was shown under the band of mature U2.3 snRNA. The radioactive signals of mature U2.3 RNAs of various genotypes were normalized to the input and compared with those of Col to obtain relative processing efficiency of various genotypes, and the value of Col was set to 1.