Discovery of a Splicing Regulator Required for Cell Cycle Progression

Abstract

In the G1 phase of the cell division cycle, eukaryotic cells prepare many of the resources necessary for a new round of growth including renewal of the transcriptional and protein synthetic capacities and building the machinery for chromosome replication. The function of G1 has an early evolutionary origin and is preserved in single and multicellular organisms, although the regulatory mechanisms conducting G1 specific functions are only understood in a few model eukaryotes. Here we describe a new G1 mutant from an ancient family of apicomplexan protozoans. Toxoplasma gondii temperature-sensitive mutant 12-109C6 conditionally arrests in the G1 phase due to a single point mutation in a novel protein containing a single RNA-recognition-motif (TgRRM1). The resulting tyrosine to asparagine amino acid change in TgRRM1 causes severe temperature instability that generates an effective null phenotype for this protein when the mutant is shifted to the restrictive temperature. Orthologs of TgRRM1 are widely conserved in diverse eukaryote lineages, and the human counterpart (RBM42) can functionally replace the missing Toxoplasma factor. Transcriptome studies demonstrate that gene expression is downregulated in the mutant at the restrictive temperature due to a severe defect in splicing that affects both cell cycle and constitutively expressed mRNAs. The interaction of TgRRM1 with factors of the tri-SNP complex (U4/U6 & U5 snRNPs) indicate this factor may be required to assemble an active spliceosome. Thus, the TgRRM1 family of proteins is an unrecognized and evolutionarily conserved class of splicing regulators. This study demonstrates investigations into diverse unicellular eukaryotes, like the Apicomplexa, have the potential to yield new insights into important mechanisms conserved across modern eukaryotic kingdoms.

Large-scale analysis of genome and transcriptome alterations in multiple tumors unveils novel cancer-relevant splicing networks

“…NUMA1 alternative splicing removes a potential threonine phosphorylation site; hence, one attractive hypothesis is that this splicing change affects NUMA1 phosphorylation, thereby impairing correct spindle positioning, leading to increased genome instability. Besides MBNL1, our analyses provided evidence for the control of NUMA1 splicing by RBM42, in agreement with its proposed role in cell cycle (Suvorova et al 2013) and pointing to an involvement of this factor in alternative splicing and cancer worth investigating further.…”

Transcript maturation in apicomplexan parasites

“…Apicomplexa introns have significant nucleotide variations at the branch point and there is an indication of additional regulatory elements located in introns and exons [22]. RNA-binding proteins of heterogeneous ribonucleoprotein (hnRNP) and serine/arginine-rich (SR) family are widely used in other eukaryotes to modulate the splice site recognition via binding to enhancer or silencer sequences [27, 28] and many of these factors are encoded in Apicomplexa genomes (Table S1) [29]. In model eukaryotes these splicing regulatory elements are known to contribute significantly to the splicing specificity, although their role in apicomplexan parasites has not yet been evaluated.…”

“…In P. falciparum , spliceosomal RNAs are abundantly expressed, modified by methylation and polyadenylation, and folded into canonical structures critical to assemble into functional snRNP complexes [26, 31, 32]. Major protein components of the spliceosome were identified by in silico analysis in several apicomplexan species [15, 29, 32, 33] (Fig. 1B, Table S1) with the level of conservation observed following the hierarchy; U5 > U4/U6 > U2 > U1 (Table S1; e-values shown in parenthesis).…”

“…Our characterization of the cell cycle mutants in Toxoplasma has identified a novel RNA-binding protein, TgRRM1, that is required for G1 phase progression in the tachyzoite [29]. Instability of the mutant TgRRM1 Y169N protein at high temperature led to immediate arrest in the early G1 phase and caused mis-splicing of nearly all intron containing genes [29].…”

“…Our characterization of the cell cycle mutants in Toxoplasma has identified a novel RNA-binding protein, TgRRM1, that is required for G1 phase progression in the tachyzoite [29]. Instability of the mutant TgRRM1 Y169N protein at high temperature led to immediate arrest in the early G1 phase and caused mis-splicing of nearly all intron containing genes [29]. The complete disruption of RNA processing inputs was unexpected as this is not observed in other eukaryotes where the competitive nature of constitutive splicing [30] and the abundance of splicing factors (80–100 th percentile RMA in Apicomplexa species, eupathDB) leads to late or random growth arrest [34–37].…”