RESUMEN
The high frequency of alternative splicing among the serine/arginine-rich (SR) family of proteins in plants has been linked to important roles in gene regulation during development and in response to environmental stress. In this article, we have searched and manually annotated all the SR proteins in the genomes of maize and sorghum. The experimental validation of gene structure by reverse transcription-polymerase chain reaction (RT-PCR) analysis revealed, with few exceptions, that SR genes produced multiple isoforms of transcripts by alternative splicing. Despite sharing high structural similarity and conserved positions of the introns, the profile of alternative splicing diverged significantly between maize and sorghum for the vast majority of SR genes. These include many transcript isoforms discovered by RT-PCR and not represented in extant expressed sequence tag (EST) collection. However, we report the occurrence of various maize and sorghum SR mRNA isoforms that display evolutionary conservation of splicing events with their homologous SR genes in Arabidopsis and moss. Our data also indicate an important role of both 5' and 3' untranslated regions in the regulation of SR gene expression. These observations have potentially important implications for the processes of evolution and adaptation of plants to land.
Asunto(s)
Empalme Alternativo/genética , Secuencia Conservada/genética , Perfilación de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Proteínas Nucleares/genética , Proteínas de Plantas/genética , Proteínas de Unión al ARN/genética , Regiones no Traducidas 3'/genética , Secuencia de Aminoácidos , Arabidopsis/genética , Bryopsida/genética , Evolución Molecular , Exones/genética , Variación Genética , Intrones/genética , Datos de Secuencia Molecular , Proteínas Nucleares/química , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/química , Homología de Secuencia de Ácido Nucleico , Factores de Empalme Serina-Arginina , Sorghum/genética , Zea mays/genéticaRESUMEN
The serine-arginine (SR)-rich splicing proteins are highly conserved RNA binding nuclear phosphor-proteins that play important roles in both regular and alternative splicing. Here we describe two novel putative SR genes from maize, designated zmRSp31A and zmRSp31B. Both genes contain characteristic RNA binding motifs RNP-1 and RNP-2, a serine/arginine-rich (RS) domain and share significant sequence similarity to the Arabidopsis atRSp31 family of SR proteins. Both zmRSp31A and zmRSp31B produce multiple transcripts by alternative splicing, of which majority of the alternatively spliced transcripts utilize non-canonical splice sites. zmRSp31A and zmRSp31B produce at least six and four transcripts, respectively, of which only one corresponds to the wild type proteins for each gene. All the alternatively spliced transcripts of both the genes, with one exception, are predicted to encode small truncated proteins containing only the RNP-2 domain of their first RNA recognition motif and completely lack the carboxyl terminal RS domain. We provide evidence that some of the alternatively spliced transcripts of both genes are associated with polysomes and interact with the translational machinery.
Asunto(s)
Empalme Alternativo/genética , Regulación de la Expresión Génica de las Plantas , Proteínas Nucleares/genética , Fosfoproteínas/genética , Empalme del ARN/genética , Proteínas de Unión al ARN/genética , Zea mays/genética , Secuencia de Aminoácidos , Proteínas de Arabidopsis/genética , Secuencia de Bases , Northern Blotting , Cartilla de ADN , Datos de Secuencia Molecular , Polirribosomas/genética , Estructura Terciaria de Proteína , Sitios de Empalme de ARN/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Alineación de Secuencia , Análisis de Secuencia de ADN , Factores de Empalme Serina-ArgininaRESUMEN
A U1-snRNP--specific 70K (U1-70K) protein is intricately involved in both constitutive and alternative splicing of pre-mRNAs. Here, we report cDNA and cognate genomic sequences of the U1-70K gene of maize and rice. The maize and rice U1-70K genes bear strong similarity to the Arabidopsis gene and each encode three transcripts in roots and shoots. Alternative splicing produces two transcripts from each gene in addition to the mRNA encoding the wild type protein. In both cases, selective inclusion of intron 6 or utilization of a cryptic donor site within intron 6 sequence generates the two alternatively spliced transcripts. This evolutionary conservation of splicing patterns between different plant species suggests an important biological function for alternative splicing in the expression of U1-70K gene.
Asunto(s)
Empalme Alternativo/genética , Secuencia Conservada/genética , Evolución Molecular , Oryza/genética , ARN Mensajero/metabolismo , Ribonucleoproteína Nuclear Pequeña U1/genética , Zea mays/genética , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , Cartilla de ADN , Datos de Secuencia Molecular , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Ribonucleoproteína Nuclear Pequeña U1/metabolismo , Alineación de Secuencia , Análisis de Secuencia de ADNRESUMEN
The propensity to capture and mobilize gene fragments by the highly abundant Helitron family of transposable elements likely impacts the evolution of genes in Zea mays. These elements provide a substrate for natural selection by giving birth to chimeric transcripts by intertwining exons of disparate genes. They also capture flanking exons by read-through transcription. Here, we describe the expression of selected Helitrons in different maize inbred lines. We recently reported that these Helitrons produce multiple isoforms of transcripts in inbred B73 via alternative splicing. Despite sharing high degrees of sequence similarity, the splicing profile of Helitrons differed among various maize inbred lines. The comparison of Helitron sequences identified unique polymorphisms in inbred B73, which potentially give rise to the alternatively spliced sites utilized by transcript isoforms. Some alterations in splicing, however, do not have obvious explanations. These observations not only add another level to the creation of transcript diversity by Helitrons among inbred lines but also provide novel insights into the cis-acting elements governing splice-site selection during pre-mRNA processing.
Asunto(s)
Elementos Transponibles de ADN/genética , Precursores del ARN/metabolismo , Zea mays/genética , Empalme Alternativo , Secuencia de Bases , Genoma de Planta , Datos de Secuencia Molecular , Raíces de Plantas/metabolismo , Brotes de la Planta/metabolismo , Precursores del ARN/genéticaRESUMEN
Helitrons are a family of mobile elements that were discovered in 2001 and are now known to exist in the entire eukaryotic kingdom. Helitrons, particularly those of maize, exhibit an intriguing property of capturing gene fragments and placing them into the mobile element. Helitron-captured genes are sometimes transcribed, giving birth to chimeric transcripts that intertwine coding regions of different captured genes. Here, we perused the B73 maize genome for high-quality, putative Helitrons that exhibit plus/minus polymorphisms and contain pieces of more than one captured gene. Selected Helitrons were monitored for expression via in silico EST analysis. Intriguingly, expression validation of selected elements by RT-PCR analysis revealed multiple transcripts not seen in the EST databases. The differing transcripts were generated by alternative selection of splice sites during pre-mRNA processing. Selection of splice sites was not random since different patterns of splicing were observed in the root and shoot tissues. In one case, an exon residing in close proximity but outside of the Helitron was found conjoined with Helitron-derived exons in the mature transcript. Hence, Helitrons have the ability to synthesize new genes not only by placing unrelated exons into common transcripts, but also by transcription readthrough and capture of nearby exons. Thus, Helitrons have a phenomenal ability to "display" new coding regions for possible selection in nature. A highly conservative, minimum estimate of the number of new transcripts expressed by Helitrons is ~11,000 or ~25% of the total number of genes in the maize genome.
Asunto(s)
Elementos Transponibles de ADN , Perfilación de la Expresión Génica , Transcriptoma , Zea mays/genética , Empalme Alternativo , Secuencia de Aminoácidos , Secuencia de Bases , Etiquetas de Secuencia Expresada/química , Regulación de la Expresión Génica de las Plantas , Orden Génico , Datos de Secuencia Molecular , Raíces de Plantas/genética , Brotes de la Planta/genética , ARN Mensajero/genética , Alineación de SecuenciaRESUMEN
We recently described a maize mutant caused by an insertion of a Helitron type transposable element (Lal, S.K., Giroux, M.J., Brendel, V., Vallejos, E. and Hannah, L.C., 2003, Plant Cell, 15: 381-391). Here we describe another Helitron insertion in the barren stalk1 gene of maize. The termini of a 6525 bp insertion in the proximal promoter region of the mutant reference allele of maize barren stalk1 gene (ba1-ref) shares striking similarity to the Helitron insertion we reported in the Shrunken-2 gene. This insertion is embedded with pseudogenes that differ from the pseudogenes discovered in the mutant Shrunken-2 insertion. Using the common terminal ends of the mutant insertions as a query, we discovered other Helitron insertions in maize BAC clones. Based on the comparison of the insertion site and PCR amplified genomic sequences, these elements inserted between AT dinucleotides. These putative non-autonomous Helitron insertions completely lacked sequences similar to RPA (replication protein A) and DNA Helicases reported in other species. A blastn analysis indicated that both the 5' and 3' termini of Helitrons are repeated in the maize genome. These data provide strong evidence that Helitron type transposable elements are active and may have played an essential role in the evolution and expansion of the maize genome.
Asunto(s)
Elementos Transponibles de ADN/genética , Seudogenes/genética , Zea mays/genética , Secuencia de Bases , Sitios de Unión/genética , Cromosomas Artificiales Bacterianos/genética , Clonación Molecular , ADN Helicasas/genética , ADN de Plantas/química , ADN de Plantas/genética , Genes de Plantas/genética , Genoma de Planta , Datos de Secuencia Molecular , Familia de Multigenes/genética , Mutagénesis Insercional , Mutación , Proteínas de Plantas/genética , Secuencias Repetitivas de Ácidos Nucleicos/genética , Alineación de Secuencia , Análisis de Secuencia de ADN , Homología de Secuencia de Ácido Nucleico , Zeína/genéticaRESUMEN
The maize mutation sh2-7527 was isolated in a conventional maize breeding program in the 1970s. Although the mutant contains foreign sequences within the gene, the mutation is not attributable to an interchromosomal exchange or to a chromosomal inversion. Hence, the mutation was caused by an insertion. Sequences at the two Sh2 borders have not been scrambled or mutated, suggesting that the insertion is not caused by a catastrophic reshuffling of the maize genome. The insertion is large, at least 12 kb, and is highly repetitive in maize. As judged by hybridization, sorghum contains only one or a few copies of the element, whereas no hybridization was seen to the Arabidopsis genome. The insertion acts from a distance to alter the splicing of the sh2 pre-mRNA. Three distinct intron-bearing maize genes were found in the insertion. Of most significance, the insertion bears striking similarity to the recently described DNA helicase-bearing transposable elements termed HELITRONS: Like Helitrons, the inserted sequence of sh2-7527 is large, lacks terminal repeats, does not duplicate host sequences, and was inserted between a host dinucleotide AT. Like Helitrons, the maize element contains 5' TC and 3' CTRR termini as well as two short palindromic sequences near the 3' terminus that potentially can form a 20-bp hairpin. Although the maize element lacks sequence information for a DNA helicase, it does contain four exons with similarity to a plant DEAD box RNA helicase. A second Helitron insertion was found in the maize genomic database. These data strongly suggest an active Helitron in the present-day maize genome.