Structural features important for the U12 snRNA binding and minor spliceosome assembly of Arabidopsis U11/U12-small nuclear ribonucleoproteins

<p>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 <i>in planta</i> 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.</p

TaRZ2 confers freezing tolerance in <i>Arabidopsis</i> plants.

<p>(A) For non cold-acclimated (NA) freezing tolerance tests, 4-week-old plants of the wild-type (WT) and TaRZ2-expressing plants (OX21, OX22, and OX23) were subjected to freezing shock at −6 for 3–6 h directly under continuous light, then transferred to normal growth conditions. (B) For cold-acclimated (CA) freezing tolerance test, 4-week-old plants were first placed at 4°C for 1 day, −1°C for 1 day, and then subjected to freezing shock at −7 for 12–25 h. Surviving plants were counted 7 days after transferring to normal growth conditions. One representative picture among repeated experiments was shown. (C) For electrolyte leakage test, leaves from the NA wild-type and TaRZ2-expresing plants were frozen at −1 to −10°C, and the extent of cellular damage was estimated by measuring electrolyte leakage.</p

Expression levels and stress-responsive expression patterns of <i>TaRZs</i> in wheat.

<p>(A) Transcript levels of <i>TaRZ1</i>, <i>TaRZ2</i>, and <i>TaRZ3</i> were analyzed via real-time RT-PCR and presented as relative expression (fold) of <i>TaRZ2</i> expression level. Two-week-old wheat plants were subjected to (B) salt, (C) dehydration, or (D) cold stress for 6, 12, 24, and 48 h, and the transcript levels of each <i>TaRZ</i> were analyzed via real-time RT-PCR and presented as the relative expression (fold) of the non-stressed controls. Values are means ± SE obtained from five independent experiments. Asterisks above each column indicate values that are statistically different from the control values (p≤0.05).</p

Comparative Functional Analysis of Wheat (<i>Triticum aestivum</i>) Zinc Finger-Containing Glycine-Rich RNA-Binding Proteins in Response to Abiotic Stresses

<div><p>Although the functional roles of zinc finger-containing glycine-rich RNA-binding proteins (RZs) have been characterized in several plant species, including <i>Arabidopsis thaliana</i> and rice (<i>Oryza sativa</i>), the physiological functions of RZs in wheat (<i>Triticum aestivum</i>) remain largely unknown. Here, the functional roles of the three wheat RZ family members, named TaRZ1, TaRZ2, and TaRZ3, were investigated using transgenic <i>Arabidopsis</i> plants under various abiotic stress conditions. Expression of <i>TaRZs</i> was markedly regulated by salt, dehydration, or cold stress. The TaRZ1 and TaRZ3 proteins were localized to the nucleus, whereas the TaRZ2 protein was localized to the nucleus, endoplasmic reticulum, and cytoplasm. Germination of all three TaRZ-expressing transgenic <i>Arabidopsis</i> seeds was retarded compared with that of wild-type seeds under salt stress conditions, whereas germination of TaRZ2- or TaRZ3-expressing transgenic <i>Arabidopsis</i> seeds was retarded under dehydration stress conditions. Seedling growth of TaRZ1-expressing transgenic plants was severely inhibited under cold or salt stress conditions, and seedling growth of TaRZ2-expressing plants was inhibited under salt stress conditions. By contrast, expression of TaRZ3 did not affect seedling growth of transgenic plants under any of the stress conditions. In addition, expression of TaRZ2 conferred freeze tolerance in <i>Arabidopsis</i>. Taken together, these results suggest that different TaRZ family members play various roles in seed germination, seedling growth, and freeze tolerance in plants under abiotic stress.</p></div

Subcellular localization of TaRZ proteins.

<p>TaRZ-GFP fusion proteins were transiently expressed in tobacco plant, and GFP signals in tobacco leaves were detected using a confocal microscope. DAPI was used to stain the nuclei, and <i>Brassica rapa</i> microsomal delta-12 fatty acid desaturase (BrFAD2) was used as a marker for ER localization. Bar  = 30 mm.</p

Maintenance of the prolonged cell division activity of AtU11/U12-31K knockdown plants.

<p>The knockdown plants (amiR1-4) survived beyond the death of the complementation lines expressing OsU11/U12-31K gene (Os31K).</p

RNA chaperone activity of OsU11/U12-31K protein.

<p>(A) The colony-forming abilities of diluted BX04 <i>E. coli</i> cells expressing either OsU11/U12-31K (Os31K), CspA (positive control), or pINIII (negative control) were examined under normal (37°C) and cold stress (20°C) conditions. (B) The DNA-melting activity of OsU11/U12-31K protein was examined by monitoring the fluorescence of a 78-nucleotide-long molecular beacon with the addition of GST-Os31K (10 µg), GST-CspA (5 µg), or GST (5 µg). (C) Enhanced RNase T₁ cleavage of the substrate RNA (R) was measured after the addition of OsU11/U12-31K protein (GST-31K). The RNase T₁-resistant bands, as indicated by arrows, disappeared in the presence of recombinant GST-31K fusion protein. The numbers above each column represent the amount of T1 added to the reaction.</p

Expression patterns of <i>U11/U12–31K</i> in <i>Arabidopsis</i> and rice.

<p>Expression patterns of <i>U11/U12-31K</i> in (A, B) Arabidopsis 20 days after germination and (C, D) rice 3 days after germination as analyzed by <i>in situ</i> hybridization. Scale bar  = 50 µm.</p

The Minor Spliceosomal Protein U11/U12-31K Is an RNA Chaperone Crucial for U12 Intron Splicing and the Development of Dicot and Monocot Plants

<div><p>U12 intron-specific spliceosomes contain U11 and U12 small nuclear ribonucleoproteins and mediate the removal of U12 introns from precursor-mRNAs. Among the several proteins unique to the U12-type spliceosomes, an <em>Arabidopsis thaliana</em> AtU11/U12-31K protein has been shown to be indispensible for proper U12 intron splicing and for normal growth and development of <em>Arabidopsis</em> plants. Here, we assessed the functional roles of the rice (<em>Oryza sativa</em>) OsU11/U12-31K protein in U12 intron splicing and development of plants. The <em>U11/U12-31K</em> transcripts were abundantly expressed in the shoot apical meristems (SAMs) of <em>Arabidopsis</em> and rice. Ectopic expression of OsU11/U12-31K in AtU11/U12-31K-defecient <em>Arabidopsis</em> mutant complemented the incorrect U12 intron splicing and abnormal development phenotypes of the <em>Arabidopsis</em> mutant plants. Impaired cell division activity in the SAMs and inflorescence stems observed in the AtU11/U12-31K-deficient mutant was completely recovered to normal by the expression of OsU11/U12-31K. Similar to <em>Arabidopsis</em> AtU11/U12-31K, rice OsU11/U12-31K was determined to harbor RNA chaperone activity. Collectively, the present findings provide evidence for the emerging idea that the U11/U12-31K protein is an indispensible RNA chaperone that functions in U12 intron splicing and is necessary for normal development of monocotyledonous plants as well as dicotyledonous plants.</p> </div

Development-dependent splicing of U12 intron-containing transcripts.

<p>The splicing patterns of U12 intron-containing transcripts were analyzed by RT-PCR in wild type (WT) and AtU11/U12-31K knockdown mutant (amiR1-4) at 3, 7, and 30 days after germination. Identical results were obtained from three independent experiments, one of which is shown.</p