Analysis of the Arabidopsis venosa4-0 mutant supports the role of VENOSA4 in dNTP metabolism.

Human Sterile alpha motif and histidine-aspartate domain containing protein 1 (SAMHD1) functions as a dNTPase to maintain dNTP pool balance. In eukaryotes, the limiting step in de novo dNTP biosynthesis is catalyzed by RIBONUCLEOTIDE REDUCTASE (RNR). In Arabidopsis, the RNR1 subunit of RNR is encoded by CRINKLED LEAVES 8 (CLS8), and RNR2 by three paralogous genes, including TSO MEANING 'UGLY' IN CHINESE 2 (TSO2). In plants, DIFFERENTIAL DEVELOPMENT OF VASCULAR ASSOCIATED CELLS 1 (DOV1) catalyzes the first step of the de novo biosynthesis of purines. Here, to explore the role of VENOSA4 (VEN4), the most likely Arabidopsis ortholog of human SAMHD1, we studied the ven4-0 point mutation, whose leaf phenotype was stronger than those of its insertional alleles. Structural predictions suggested that the E249L substitution in the mutated VEN4-0 protein rigidifies its 3D structure. The morphological phenotypes of the ven4, cls8, and dov1 single mutants were similar, and those of the ven4 tso2 and ven4 dov1 double mutants were synergistic. The ven4-0 mutant had reduced levels of four amino acids related to dNTP biosynthesis, including glutamine and glycine, which are precursors in the de novo purine biosynthesis. Our results reveal high functional conservation between VEN4 and SAMHD1 in dNTP metabolism.

Missplicing suppressor alleles of Arabidopsis PRE-MRNA PROCESSING FACTOR 8 increase splicing fidelity by reducing the use of novel splice sites.

Efficient splicing requires a balance between high-fidelity splice-site (SS) selection and speed. In Saccharomyces cerevisiae, Pre-mRNA processing factor 8 (Prp8) helps to balance precise SS selection and rapid, efficient intron excision and exon joining. argonaute1-52 (ago1-52) and incurvata13 (icu13) are hypomorphic alleles of the Arabidopsis thaliana genes ARGONAUTE1 (AGO1) and AUXIN RESISTANT6 (AXR6) that harbor point mutations creating a novel 3'SS and 5'SS, respectively. The spliceosome recognizes these novel SSs, as well as the intact genuine SSs, producing a mixture of wild-type and aberrant mature mRNAs. Here, we characterized five novel mutant alleles of PRP8 (one of the two Arabidopsis co-orthologs of yeast Prp8), naming these alleles morphology of ago1-52 suppressed5 (mas5). In the mas5-1 background, the spliceosome preferentially recognizes the intact genuine 3'SS of ago1-52 and 5'SS of icu13. Since point mutations that damage genuine SSs make the spliceosome prone to recognizing cryptic SSs, we also tested alleles of four genes carrying damaged genuine SSs, finding that mas5-1 did not suppress their missplicing. The mas5-1 and mas5-3 mutations represent a novel class of missplicing suppressors that increase splicing fidelity by hampering the use of novel SSs, but do not alter general pre-mRNA splicing.

SMALL ORGAN4 Is a Ribosome Biogenesis Factor Involved in 5.8S Ribosomal RNA Maturation.

Ribosome biogenesis is crucial for cellular metabolism and has important implications for disease and aging. Human (Homo sapiens) glioma tumor-suppressor candidate region gene2 (GLTSCR2) and yeast (Saccharomyces cerevisiae) Nucleolar protein53 (Nop53) are orthologous proteins with demonstrated roles as ribosome biogenesis factors; knockdown of GLTSCR2 impairs maturation of 18S and 5.8S ribosomal RNAs (rRNAs), and Nop53 is required for maturation of 5.8S and 25S rRNAs. Here, we characterized SMALL ORGAN4 (SMO4), the most likely ortholog of human GLTSCR2 and yeast Nop53 in Arabidopsis (Arabidopsis thaliana). Loss of function of SMO4 results in a mild morphological phenotype; however, we found that smo4 mutants exhibit strong cytological and molecular phenotypes: nucleolar hypertrophy and disorganization, overaccumulation of 5.8S and 18S rRNA precursors, and an imbalanced 40S:60S ribosome subunit ratio. Like yeast Nop53 and human GLTSCR2, Arabidopsis SMO4 participates in 5.8S rRNA maturation. In yeast, Nop53 cooperates with mRNA transport4 (Mtr4) for 5.8S rRNA maturation. In Arabidopsis, we found that SMO4 plays similar roles in the 5.8S rRNA maturation pathway than those described for MTR4. However, SMO4 seems not to participate in the degradation of by-products derived from the 5'-external transcribed spacer (ETS) of 45S pre-rRNA, as MTR4 does.