SERRATE Protein Research Articles

The SERRATE protein is involved in alternative splicing in Arabidopsis thaliana.

How alternative splicing (AS) is regulated in plants has not yet been elucidated. Previously, we have shown that the nuclear cap-binding protein complex (AtCBC) is involved in AS in Arabidopsis thaliana. Here we show that both subunits of AtCBC (AtCBP20 and AtCBP80) interact with SERRATE (AtSE), a protein involved in the microRNA biogenesis pathway. Moreover, using a high-resolution reverse transcriptase-polymerase chain reaction AS system we have found that AtSE influences AS in a similar way to the cap-binding complex (CBC), preferentially affecting selection of 5' splice site of first introns. The AtSE protein acts in cooperation with AtCBC: many changes observed in the mutant lacking the correct SERRATE activity were common to those observed in the cbp mutants. Interestingly, significant changes in AS of some genes were also observed in other mutants of plant microRNA biogenesis pathway, hyl1-2 and dcl1-7, but a majority of them did not correspond to the changes observed in the se-1 mutant. Thus, the role of SERRATE in AS regulation is distinct from that of HYL1 and DCL1, and is similar to the regulation of AS in which CBC is involved.

Active 5' splice sites regulate the biogenesis efficiency of Arabidopsis microRNAs derived from intron-containing genes.

Arabidopsis, miR402 that is encoded within the first intron of a protein-coding gene At1g77230, is induced by heat stress. Its upregulation correlates with splicing inhibition and intronic proximal polyA site selection. It suggests that miR402 is not processed from an intron, but rather from a shorter transcript after selection of the proximal polyA site within this intron. Recently, introns and active 5΄ splice sites (5΄ss’) have been shown to stimulate the accumulation of miRNAs encoded within the first exons of intron-containing MIR genes. In contrast, we have observed the opposite effect of splicing inhibition on intronic miR402 production. Transient expression experiments performed in tobacco leaves revealed a significant accumulation of the intronic mature miR402 when the 5΄ss of the miR402-hosting intron was inactivated. In contrast, when the miR402 stem-loop structure was moved into the first exon, mutation of the first-intron 5΄ss resulted in a decrease in the miRNA level. Thus, the 5΄ss controls the efficiency of miRNA biogenesis. We also show that the SERRATE protein (a key component of the plant microprocessor) colocalizes and interacts with several U1 snRNP auxiliary proteins. We postulate that SERRATE-spliceosome connections have a direct effect on miRNA maturation.

Functional mapping of the plant small RNA methyltransferase: HEN1 physically interacts with HYL1 and DICER-LIKE 1 proteins.

Methylation of 3′-terminal nucleotides of miRNA/miRNA* is part of miRNAs biogenesis in plants but is not found in animals. In Arabidopsis thaliana this reaction is carried out by a multidomain AdoMet-dependent 2′-O-methyltransferase HEN1. Using deletion and structure-guided mutational analysis, we show that the double-stranded RNA-binding domains R1 and R2 of HEN1 make significant but uneven contributions to substrate RNA binding, and map residues in each domain responsible for this function. Using GST pull-down assays and yeast two-hybrid analysis we demonstrate direct HEN1 interactions, mediated by its FK506-binding protein-like domain and R2 domain, with the microRNA biogenesis protein HYL1. Furthermore, we find that HEN1 forms a complex with DICER-LIKE 1 (DCL1) ribonuclease, another key protein involved in miRNA biogenesis machinery. In contrast, no direct interaction is detectable between HEN1 and SERRATE. On the basis of these findings, we propose a mechanism of plant miRNA maturation which involves binding of the HEN1 methyltransferase to the DCL1•HYL1•miRNA complex excluding the SERRATE protein.

The SERRATE protein is involved in alternative splicing in Arabidopsis thaliana

How alternative splicing (AS) is regulated in plants has not yet been elucidated. Previously, we have shown that the nuclear cap-binding protein complex (AtCBC) is involved in AS in Arabidopsis thaliana. Here we show that both subunits of AtCBC (AtCBP20 and AtCBP80) interact with SERRATE (AtSE), a protein involved in the microRNA biogenesis pathway. Moreover, using a high-resolution reverse transcriptase-polymerase chain reaction AS system we have found that AtSE influences AS in a similar way to the cap-binding complex (CBC), preferentially affecting selection of 5′ splice site of first introns. The AtSE protein acts in cooperation with AtCBC: many changes observed in the mutant lacking the correct SERRATE activity were common to those observed in the cbp mutants. Interestingly, significant changes in AS of some genes were also observed in other mutants of plant microRNA biogenesis pathway, hyl1-2 and dcl1-7, but a majority of them did not correspond to the changes observed in the se-1 mutant. Thus, the role of SERRATE in AS regulation is distinct from that of HYL1 and DCL1, and is similar to the regulation of AS in which CBC is involved.

Bunched sets a boundary for Notch signaling to pattern anterior eggshell structures during Drosophila oogenesis

Organized boundaries between different cell fates are critical in patterning and organogenesis. In some tissues, long-range signals position a boundary, and local Notch signaling maintains it. How Notch activity is restricted to boundary regions is not well understood. During Drosophila oogenesis, the long-range signals EGF and Dpp regulate expression of bunched ( bun), which encodes a homolog of mammalian transcription factors TSC-22 and GILZ. Here, we show that bun establishes a boundary for Notch signaling in the follicle cell epithelium. Notch signaling is active in anterior follicle cells and is required for concurrent follicle cell reorganizations including centripetal migration and operculum formation. bun is required in posterior columnar follicle cells to repress the centripetal migration fate, including gene expression, cell shape changes and accumulation of cytoskeletal components. bun mutant clones adjacent to the centripetally migrating follicle cells showed ectopic Notch responses. bun is necessary, but not sufficient, to down-regulate Serrate protein levels throughout the follicular epithelium. These data indicate that Notch signaling is necessary, but not sufficient, for centripetal migration and that bun regulates the level of Notch stimulation to position the boundary between centripetally migrating and stationary columnar follicle cells.

Positive and negative control of Serrate expression during early development of the Drosophila wing

The product of the Drosophila gene Serrate acts as a short-range signal during wing development to induce the organising centre at the dorsal/ventral compartment boundary, from which growth and patterning of the wing is controlled. Regulatory elements reflecting the early Serrate expression in the dorsal compartment of the wing disc have recently been confined to a genomic fragment in the 5′-upstream region of the gene. Here we present data to suggest that this fragment responds to various positive and negative inputs required for the early Serrate expression. First, activation and maintenance of expression in the dorsal compartment of the wing discs of second and early third instar larvae depends on apterous, as revealed by reporter gene expression in discs either lacking or ectopically expressing apterous. Second, transcriptional downregulation during third larval instar is mediated by hiiragi. Finally, this regulatory element responds to Delta signalling in a nonautonomous way to maintain Serrate expression along the dorsal margin. The results clearly show that some of the previously described transactivators of Serrate protein expression, e.g. fringe, act on elements required for later aspects of Serrate expression.

A dominant-negative form of Serrate acts as a general antagonist of Notch activation.

Specification of the dorsal-ventral compartment boundary in the developing Drosophila wing disc requires activation of NOTCH from its dorsal ligand SERRATE and its ventral ligand DELTA. Both NOTCH ligands are required in this process and one cannot be substituted for the other. In the wing disc, expression of a dominant-negative, truncated form of SERRATE called BD(G), is capable of inhibiting NOTCH activation in the ventral but not the dorsal compartments. We demonstrate that BD(G) can act as a general antagonist of both SERRATE and DELTA mediated NOTCH interactions, however, BD(G) retains the SERRATE protein domain targeted by FRINGE, hence its antagonistic effects are restricted in the dorsal wing disc. Our findings suggest a model in which ligand binding to NOTCH is a necessary but insufficient step toward NOTCH activation.

Fringe modulates Notch-ligand interactions.

The Notch family of transmembrane receptor proteins mediate developmental cell-fate decisions, and mutations in mammalian Notch genes have been implicated in leukaemia, breast cancer, stroke and dementia. During wing development in Drosophila, the Notch receptor is activated along the border between dorsal and ventral cells, leading to the specification of specialized cells that express Wingless (Wg) and organize wing growth and patterning. Three genes, fringe (fng), Serrate (Ser) and Delta (Dl), are involved in the cellular interactions leading to Notch activation. Ser and Dl encode transmembrane ligands for Notch, whereas fng encodes a pioneer protein. We have investigated the relationship between these genes by a combination of expression and coexpression studies in the Drosophila wing. We found that Ser and Dl maintain each other's expression by a positive feedback loop. fng is expressed specifically by dorsal cells and functions to position and restrict this feedback loop to the developing dorsal-ventral boundary. This is achieved by fng through a cell-autonomous mechanism that inhibits a cell's ability to respond to Serrate protein and potentiates its ability to respond to Delta protein.

Phenotypic and molecular characterization of SerD, a dominant allele of the Drosophila gene Serrate.

The Drosophila gene Serrate (Ser) encodes a transmembrane protein with 14 epidermal growth factor--like repeats in its extracellular domain, which is required for the control of cell proliferation and pattern formation during wing development. Flies hetero- or homozygous for the dominant mutation SerD exhibit scalloping of the wing margin due to cell death during pupal stages. SerD is associated with an insertion of the transposable element Tirant in the 3' untranslated region of the gene, resulting in the truncation of the Ser RNA, thereby eliminating putative RNA degradation signals located further downstream. This leads to increased stability of Ser RNA and higher levels of Serrate protein. In wing discs of wild-type third instar larvae, the Serrate protein exhibits a complex expression pattern, including a strong stripe dorsal and a weaker stripe ventral to the prospective wing margin. Wing discs of SerD third instar larvae exhibit additional Serrate protein expression in the edge zone of the future wing margin, where it is normally not detectable. In these cells expression of wing margin specific genes, such as cut and wingless, is repressed. By using the yeast Gal4 system to induce locally restricted ectopic expression of Serrate in the edge zone of the prospective wing margin, we can reproduce all aspects of the SerD wing phenotype, that is, repression of wing margin-specific genes, scalloping of the wing margin and enhancement of the Notch haplo-insufficiency wing phenotype. This suggests that expression of the Serrate protein in the cells of the edge zone of the wing margin, where it is normally absent, interferes with the proper development of the margin.