RNA BIOLOGY 2016, VOL. 13, NO. 7, 670–679 http://dx.doi.org/10.1080/15476286.2016.1191736
RESEARCH PAPER
Structural features important for the U12 snRNA binding and minor spliceosome assembly of Arabidopsis U11/U12-small nuclear ribonucleoproteins Su Jung Parka,*, Hyun Ju Junga,*, Sy Nguyen Dinha, and Hunseung Kanga a
Department of Plant Biotechnology, College of Agriculture and Life Sciences, Chonnam National University, Yongbong-dong, Buk-gu, Gwangju, South Korea
ABSTRACT
ARTICLE HISTORY
Although seven proteins unique to U12 intron-specific minor spliceosomes, denoted as U11/U12-65K, -59K, -48K, -35K, -31K, -25K, and -20K, have been identified in humans and the roles of some of them have been demonstrated, the functional role of most of these proteins in plants is not understood. A recent study demonstrated that Arabidopsis U11/U12-65K is essential for U12 intron splicing and normal plant development. However, the structural features and sequence motifs important for 65 K binding to U12 snRNA and other spliceosomal proteins remain unclear. Here, we demonstrated by domain-deletion analysis that the C-terminal region of the 65 K protein bound specifically to the stem-loop III of U12 snRNA, whereas the N-terminal region of the 65 K protein was responsible for interacting with the 59 K protein. Analysis of the interactions between each snRNP protein using yeast two-hybrid analysis and in planta bimolecular fluorescence complementation and luciferase complementation imaging assays demonstrated that the core interactions among the 65 K, 59 K, and 48 K proteins were conserved between plants and animals, and multiple interactions were observed among the U11/U12-snRNP proteins. Taken together, these results reveal that U11/U12-65K is an indispensible component of the minor spliceosome complex by binding to both U11/U12-59K and U12 snRNA, and that multiple interactions among the U11/ U12-snRNP proteins are necessary for minor spliceosome assembly.
Received 12 February 2016 Revised 13 May 2016 Accepted 13 May 2016
Introduction Eukaryotic precursor (pre)-mRNA splicing is an important RNA metabolism necessary for regulated gene expression, in which introns (non-coding sequences) are removed from the pre-mRNAs by two distinct spliceosome complexes. Majority of introns are U2-type, and splicing of the U2-type introns is mediated by the major spliceosome.1,2 U12-type introns are relatively rare comprising
RESEARCH PAPER
Structural features important for the U12 snRNA binding and minor spliceosome assembly of Arabidopsis U11/U12-small nuclear ribonucleoproteins Su Jung Parka,*, Hyun Ju Junga,*, Sy Nguyen Dinha, and Hunseung Kanga a
Department of Plant Biotechnology, College of Agriculture and Life Sciences, Chonnam National University, Yongbong-dong, Buk-gu, Gwangju, South Korea
ABSTRACT
ARTICLE HISTORY
Although seven proteins unique to U12 intron-specific minor spliceosomes, denoted as U11/U12-65K, -59K, -48K, -35K, -31K, -25K, and -20K, have been identified in humans and the roles of some of them have been demonstrated, the functional role of most of these proteins in plants is not understood. A recent study demonstrated that Arabidopsis U11/U12-65K is essential for U12 intron splicing and normal plant development. However, the structural features and sequence motifs important for 65 K binding to U12 snRNA and other spliceosomal proteins remain unclear. Here, we demonstrated by domain-deletion analysis that the C-terminal region of the 65 K protein bound specifically to the stem-loop III of U12 snRNA, whereas the N-terminal region of the 65 K protein was responsible for interacting with the 59 K protein. Analysis of the interactions between each snRNP protein using yeast two-hybrid analysis and in planta bimolecular fluorescence complementation and luciferase complementation imaging assays demonstrated that the core interactions among the 65 K, 59 K, and 48 K proteins were conserved between plants and animals, and multiple interactions were observed among the U11/U12-snRNP proteins. Taken together, these results reveal that U11/U12-65K is an indispensible component of the minor spliceosome complex by binding to both U11/U12-59K and U12 snRNA, and that multiple interactions among the U11/ U12-snRNP proteins are necessary for minor spliceosome assembly.
Received 12 February 2016 Revised 13 May 2016 Accepted 13 May 2016
Introduction Eukaryotic precursor (pre)-mRNA splicing is an important RNA metabolism necessary for regulated gene expression, in which introns (non-coding sequences) are removed from the pre-mRNAs by two distinct spliceosome complexes. Majority of introns are U2-type, and splicing of the U2-type introns is mediated by the major spliceosome.1,2 U12-type introns are relatively rare comprising
