Abstract
Ubiquitin-like proteins play important roles in the regulation of many biological processes. UBL5 (Ubiquitin-like protein 5)/Hub1 (Homologous to ubiquitin 1), a member of the ubiquitin family, acts as a ubiquitin-like modifier on a specific target, the spliceosomal protein Snu66, in yeast and human cells. The 22nd aspartic acid (Asp22) is involved in the attachment of Hub1 to the Hub1 interaction domain (HIND) of Snu66 in yeast to modulate spliceosomal activity. Hub1 differs from other modifiers which interact covalently with their targets. It modulates pre-mRNA splicing by binding to Snu66 non-covalently in both yeast and human cells. However, the molecular mechanisms of Hub1-mediated pre-mRNA splicing in plant systems remains unclear. To better understand the function of Hub1 in plants, we examined the role of this ubiquitin-like modifier in Arabidopsis thaliana, which has two Hub1 homologues. Arabidopsis UBL5/Hub1(UBL5) is highly conserved at the amino acid level, compared to eukaryotic homologues in both plants and animals. In this study, phenotypic analysis of A. thaliana with reduced UBL5 gene expression, generated by RNA interference of AtUBL5a and AtUBL5b were performed. Interestingly, knock down plants of AtUBL5 showed abnormalities in root elongation, plant development, and auxin response. AtUBL5b is highly expressed in the vascular tissue of the leaf, stem, and root tissue. Yeast two-hybrid analysis revealed that AtUBL5a and AtUBL5b interact with the putative splicing factor AtPRP38 through its C-terminal domain (AtPRP38C). Knock down of AtUBL5b resulted in a pattern of insufficient pre-mRNA splicing in several introns of AtCDC2, and in introns of IAA1, IAA4, and IAA5. Defects of pre-mRNA splicing in an AtPRP38 mutant resulted in an insufficient pre-mRNA splicing pattern in the intron of IAA1. Based on these results, we showed that AtUBL5b positively regulates plant root elongation and development through pre-mRNA splicing with AtPRP38C in A. thaliana.
Highlights
Ubiquitin and ubiquitin-like proteins (UBLs) are multifunctional regulatory modifiers of eukaryotic and eubacterial proteins [1]
We demonstrated that AtUBL5 interacts with the Hub1 interaction domain (HIND) of AtPRP38C, and regulates root elongation and development by modulating pre-mRNA splicing in AtUBL5 knockdown A. thaliana
There are two yeast Hub1 homologues in the genome of A. thaliana genome, which are designated as AtUBL5a (At3g45180) and AtUBL5b (At5g42300) (Fig 1A)
Summary
Ubiquitin and ubiquitin-like proteins (UBLs) are multifunctional regulatory modifiers of eukaryotic and eubacterial proteins [1]. Research on yeast and human homologous to ubiquitin 1 (Hub, known as Ubl5) protein led to the discovery of Hub1-dependent splicing processes [2,3,4,5,6,7]. Hub binding to S. cerevisiae Snu (ScSnu66) affects the interaction with spliceosomal proteins and splicing [5]. Hub has important functions as a modulator of spliceosome activity and a facilitator for alternative splicing in both animal and yeast cells [2]. The C-terminal domain of Hub is not important for target binding; rather, it is the 22nd aspartic acid (Asp22) that is involved in the attachment of Hub to the HIND of Snu in yeast and human cells [2, 4,5,6,7]. Hub is homologous to ubiquitin-like modifiers, it acts via a unique mechanism and represents a distinctive functional ubiquitin-like modifier system
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