Biology subjects

Sarmiento-Manus, R.

Publications and source records attributed to Sarmiento-Manus, R..

5 recordsLinked to original sources

bioRxiv2024

CXIP4 depletion causes early lethality and pre-mRNA missplicing in Arabidopsis

Zinc knuckle (ZCCHC) motif-containing proteins are present in unicellular and multicellular eukaryotes and most ZCCHC proteins with known functions participate in the metabolism of various classes of RNA, such as mRNAs, ribosomal RNAs, and microRNAs. The Arabidopsis (Arabidopsis thaliana) genome encodes 69 ZCCHC-containing proteins, but the functions of most remain unclear. One of these proteins is CAX-INTERACTING PROTEIN 4 (CXIP4), which has been classified as a PTHR31437 family member, along with human SREK1-interacting protein 1 (SREK1IP1), which is thought to function in pre-mRNA splicing and RNA methylation. Metazoan SREK1IP1-like and plant CXIP4-like proteins only share a ZCCHC motif, and their functions remain almost entirely unknown. We studied two loss-of-function alleles of Arabidopsis CXIP4, the first mutations in PTHR31437 family genes described to date: cxip4-1 is likely null and shows early lethality, and cxip4-2 is hypomorphic and viable, with pleiotropic morphological defects. The cxip4-2 mutant exhibited deregulation of defense genes and upregulation of transcription factor encoding genes, some of which might explain its developmental defects. This mutant also exhibited increased intron retention events, and the specific functions of misspliced genes, such as those involved in "gene silencing by DNA methylation" and "mRNA polyadenylation factor" suggest that CXIP4 has additional functions. The CXIP4 protein localizes to the nucleus in a pattern resembling nuclear speckles, which are rich in splicing factors. Therefore, CXIP4 is required for plant survival and proper development, and mRNA maturation.

plant biology↗

bioRxiv2024

Functional conservation and divergence of Arabidopsis VENOSA4 and human SAMHD1 in DNA repair

The human deoxyribonucleoside triphosphatase (dNTPase) Sterile alpha motif and histidine-aspartate domain containing protein 1 (SAMHD1) has a dNTPase-independent role in repairing DNA double-strand breaks (DSBs) by homologous recombination (HR). Here, we show that VENOSA4 (VEN4), the probable Arabidopsis thaliana ortholog of SAMHD1, also functions in DSB repair by HR. The ven4 loss-of-function mutants showed increased DNA ploidy and deregulated DNA repair genes, suggesting DNA damage accumulation. Hydroxyurea, which blocks DNA replication and generates DSBs, induced VEN4 expression. The ven4 mutants were hypersensitive to hydroxyurea, with decreased DSB repair by HR. Metabolomic analysis of the strong ven4-0 mutant revealed depletion of metabolites associated with DNA damage responses. In contrast to SAMHD1, VEN4 showed no evident involvement in preventing R-loop accumulation. Our study thus reveals functional conservation in DNA repair by VEN4 and SAMHD1. One sentence summaryHuman SAMHD1 is involved in dNTP metabolism and DNA repair; the latter function is conserved in VEN4, its likely Arabidopsis ortholog.

plant biology↗

bioRxiv2024

Postembryonic developmental roles of the Arabidopsis KEULE gene

Cytokinesis in plant cells begins with the fusion of vesicles that transport cell wall materials at the center of the cell division plane, where the cell plate forms and expands radially until it fuses with the parental cell wall at the preprophase band. Vesicle fusion is facilitated by trans-SNARE complexes, with assistance from Sec1/Munc18 (SM) proteins. The SNARE protein KNOLLE and the SM protein KEULE are required for membrane fusion at the cell plate. Due to the crucial function of KEULE, all Arabidopsis (Arabidopsis thaliana) keule mutants identified to date are seedling lethal. Here, we identified the Arabidopsis serrata4-1 (sea4-1) and sea4-2 mutants, which carry recessive, hypomorphic alleles of KEULE. Homozygous sea4-1 and sea4-2 plants are viable and fertile but exhibit smaller rosettes and fewer leaves at bolting than the wild type. Their leaves are serrated, small, and undulated, with a complex venation pattern, develop necrotic patches, and undergo premature senescence. We established a likely relationship between these phenotypes and their defects in cytokinesis through reduced cell wall integrity and increased unfolded protein response. These findings shed light on the roles of KEULE in postembryonic development, particularly in the patterning of rosette leaves and leaf margins.

plant biology↗

bioRxiv2023

Cross-kingdom conservation of Arabidopsis RPS24 function in 18S rRNA maturation

All 81 ribosomal proteins (RPs) that form the Arabidopsis (Arabidopsis thaliana) 80S ribosome are encoded by several paralogous genes. For example, the nearly identical RPS24A and RPS24B proteins are encoded by RPS24A and RPS24B, respectively. Here we explored the functions of RPS24A and RPS24B in Arabidopsis. Their encoding genes exhibit combined haploinsufficiency, as at least two wild-type copies of either RPS24A or RPS24B are required for plant viability and at least three are required for normal plant development. Loss-of-function of either gene caused a pointed-leaf phenotype, a typical phenotype of null or hypomorphic recessive alleles of genes encoding ribosome biogenesis factors (RBFs) or RPs. We also found that RPS24A and RPS24B act as RBFs during early stages of 18S ribosomal RNA (rRNA) maturation, as loss of RPS24A or RPS24B function reduced the 18S/25S rRNA ratio. An RPS24B-GFP fusion protein predominantly localized to the nucleolus, as expected. The rps24b-2 mutation strengthened the phenotypes of the RBF mutants mRNA transporter4-2 and small organ4-3, which are defective in 5.8S rRNA maturation. This synergistic interaction might be an effect of increased 45S rDNA transcription, which we also observed in the rps24 mutants. Therefore, the Arabidopsis RPS24 proteins act as RBFs during 18S rRNA maturation, like their human and yeast putative orthologs. Only two plant RPs were previously shown to act not only as structural components of the ribosome but also as RBFs. We provide evidence that RPS24 proteins also regulate 45S rDNA transcription, which has not been described for their yeast or human orthologs.

plant biology↗

bioRxiv2023

Analysis of Arabidopsis venosa4-0 supports the role of VENOSA4 in dNTP homeostasis

An imbalance in the deoxyribonucleoside triphosphate (dNTP) pool caused by an increase or decrease in the levels of any of the four dNTPs leads to increased DNA mutations, overloading DNA repair mechanisms. The human protein SAMHD1 (Sterile alpha motif and histidine-aspartate domain containing protein 1) functions as a dNTPase to maintain the balance of the dNTP pool, as well as in DNA repair. In eukaryotes, the limiting step in de novo dNTP synthesis is catalyzed by RIBONUCLEOTIDE REDUCTASE (RNR), which consists of two R1 and two R2 subunits. In Arabidopsis, RNR1 is encoded by CRINKLED LEAVES 8 (CLS8) and RNR2 by three paralogous genes, including TSO2 (TSO MEANING UGLY IN CHINESE 2). In plants, the de novo biosynthesis of purines occurs within the chloroplast, and DOV1 (DIFFERENTIAL DEVELOPMENT OF VASCULAR ASSOCIATED CELLS 1) catalyzes the first step of this pathway. Here, to explore the role of VENOSA4 (VEN4), the most likely Arabidopsis ortholog of human SAMHD1, we studied the ven4-0 mutant. The mutant leaf phenotype caused by the ven4-0 point mutation was stronger than those of T-DNA insertional ven4 mutations. Structural predictions suggested that the E249L amino acid substitution in the mutated VEN4-0 protein rigidifies its 3D structure compared to wild-type VEN4. 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 pathway. Finally, despite its annotation in some databases, At5g40290, a paralog of VEN4, is likely a pseudogene. These observations support the previously proposed role of VEN4 in dNTP metabolism. Our results reveal a high degree of cross-kingdom functional conservation between VEN4 and SAMHD1 in dNTP homeostasis.

plant biology↗