The Spliceosome-Activating Complex: Molecular Mechanisms Underlying the Function of a Pleiotropic Regulator

Abstract

Correct interpretation of the coding capacity of RNA polymerase II transcribed eukaryotic genes is determined by the recognition and removal of intronic sequences of pre-mRNAs by the spliceosome. Our current knowledge on dynamic assembly and subunit interactions of the spliceosome mostly derived from the characterization of yeast, Drosophila, and human spliceosomal complexes formed on model pre-mRNA templates in cell extracts. In addition to sequential structural rearrangements catalyzed by ATP-dependent DExH/D-box RNA helicases, catalytic activation of the spliceosome is critically dependent on its association with the NineTeen Complex (NTC) named after its core E3 ubiquitin ligase subunit PRP19. NTC, isolated recently from Arabidopsis, occurs in a complex with the essential RNA helicase and GTPase subunits of the U5 small nuclear RNA particle that are required for both transesterification reactions of splicing. A compilation of mass spectrometry data available on the composition of NTC and spliceosome complexes purified from different organisms indicates that about half of their conserved homologs are encoded by duplicated genes in Arabidopsis. Thus, while mutations of single genes encoding essential spliceosome and NTC components lead to cell death in other organisms, differential regulation of some of their functionally redundant Arabidopsis homologs permits the isolation of partial loss of function mutations. Non-lethal pleiotropic defects of these mutations provide a unique means for studying the roles of NTC in co-transcriptional assembly of the spliceosome and its crosstalk with DNA repair and cell death signaling pathways.

Recruitment of the NineTeen Complex to the activated spliceosome requires AtPRMT5

“…Intriguingly, among the proteins that interact with Prp8, our MS results identified a set of splicing factors, which form the Prp19 complex (Prp19C), also known as NTC (21,31,32). Compared with Col, the atprmt5 mutants showed significantly reduced normalized spectral counts from the Prp19C/NTC core proteins, including MAC3A and MAC3B (human Prp19 homolog in Arabidopsis), MOS4 (human SPF27 homolog in Arabidopsis), CDC5, and PRL1, and the Prp19C/NTC-associated proteins (SYF1, AQR, CRN1c, SKIP, ECM2, ISY1, and PPI) (Fig.…”

Role for LSM genes in the regulation of circadian rhythms

“…To identify connections between the circadian clock and AS in plants, we investigated which splicing factors were clock-regulated (24)(25)(26). We found 83 splicing-related genes that cycle at the mRNA level, and thus represent candidate regulators of clock-mediated AS (Dataset S1).…”

Inactivation of Plasma Membrane–Localized CDPK-RELATED KINASE5 Decelerates PIN2 Exocytosis and Root Gravitropic Response inArabidopsis

“…CRK5 (called alternatively PIP-D) was originally identified in yeast two-hybrid and in vitro protein binding assays as a potential interaction partner of the C-terminal WD40-repeat domain of nuclear PRL1 protein, a subunit of the spliceosome-activating complex (Németh et al, 1998;Koncz et al, 2012). Although subsequent studies did not confirm in vivo formation of a CRK5-PRL1 complex under normal growth conditions, we found that inactivation of the CRK5 gene by a T-DNA insertion in the MPIZ mutant line 38225 (crk5-1; Ríos et al, 2002; see Supplemental Figure 1A online) resulted in reduced primary root elongation ( Figure 1A), analogously to the prl1 mutation (Németh et al, 1998).…”