<i>Arabidopsis</i> AtRRP44A Is the Functional Homolog of Rrp44/Dis3, an Exosome Component, Is Essential for Viability and Is Required for RNA Processing and Degradation

<div><p>The RNA exosome is a multi-subunit complex that is responsible for 3ʹ to 5ʹ degradation and processing of cellular RNA. Rrp44/Dis3 is the catalytic center of the exosome in yeast and humans. However, the role of Rrp44/Dis3 homologs in plants is still unidentified. Here, we show that <i>Arabidopsis</i> AtRRP44A is the functional homolog of Rrp44/Dis3, is essential for plant viability and is required for RNA processing and degradation. We characterized AtRRP44A and AtRRP44B/SOV, two predicted <i>Arabidopsis</i> Rrp44/Dis3 homologs. AtRRP44A could functionally replace <i>S. cerevisiae</i> Rrp44/Dis3, but AtRRP44B/SOV could not. <i>rrp44a</i> knock-down mutants showed typical phenotypes of exosome function deficiency, 5.8S rRNA 3ʹ extension and rRNA maturation by-product over-accumulation, but <i>rrp44b</i> mutants did not. Conversely, AtRRP44B/SOV mutants showed elevated levels of a selected mRNA, on which <i>rrp44a</i> did not have detectable effects. Although T-DNA insertion mutants of AtRRP44B/SOV had no obvious phenotype, those of AtRRP44A showed defects in female gametophyte development and early embryogenesis. These results indicate that AtRRP44A and AtRRP44B/SOV have independent roles for RNA turnover in plants.</p> </div

Model for the roles of <i>A. thaliana</i> AtRRP44A and AtRRP44B/SOV in RNA processing and degradation.

<p>AtRRP44A localizes to the nucleus and processes rRNAs with the exosome complex. However, AtRRP44B/SOV localizes to the cytoplasm and targets a select subset of mRNAs.</p

Analysis of rRNA processing and degradation.

<p>(A) Diagram illustrating the 5.8S rRNA processing intermediates and the rRNA maturation by-product generated from the 5ʹ ETS (P-Pʹ) compared with the 35S precursor [27]. Horizontal red arrows represent the positions of oligonucleotide probes used in this study. (B) The 5.8S rRNA 3ʹ extension is processed by AtRRP44A, AtRRP4 and AtRRP41, but not AtRRP44B/SOV. (C) The 5ʹ ETS is degraded by AtRRP44A, AtRRP4 and AtRRP41, but not AtRRP44B/SOV. RNA gel blots of 5.8S rRNA precursors (B) or the 5ʹ ETS (C). Total RNAs were isolated from 10 dpg rosette leaves of Col-0 (wild type: WT), <i>gusKD-2</i> (VC), <i>rrp44aKD-1</i>, <i>rrp44aKD-2</i>, <i>rrp44b-1</i>, <i>rrp44b-2</i> and <i>mtr4-1</i> plants or from <i>gusKD-2</i>, <i>rrp4KD-3</i>, <i>rrp41KD-1</i> and <i>rrp44aKD-1</i> plants (B and C). <i>mtr4-1</i> was used to determine the sequence of 5.8S processing intermediates [27]. Total RNAs were separated on 6% polyacrylamide gels. Methylene blue staining of 5S rRNA is shown as a loading control. Relative RNA levels estimated from band signals are indicated at the bottom of each lane as mean values ± SE with RNA levels in Col-0 plants set to 1.0.. Values for which P<0.05 (Tukey’s test) compared to corresponding wild type plants (<i>gusKD-2</i> or Col-0) were shown in red. Two (B and C: Left panels) or three (B and C: Right panels) biological replicates were performed for all RNA gel blots. </p

<i>Arabidopsis</i> Rrp44/Dis3 homologs.

<p>(A) Schematics of the Rrp44/Dis3 homologs <i>S. cerevisiae</i> Rrp44 (ScRrp44), human RRP44/DIS3 (hRRP44), and <i>A. thaliana</i> AtRRP44A and AtRRP44B/SOV. Yellow and blue boxes represent the PIN and RNB domains, respectively, that are conserved among Rrp44/Dis3 homologs. aa represents amino acids. (B) AtRRP44A complements the S. <i>cerevisiae </i><i>rrp44</i> doxycycline (DOX) repressible mutant. Growth phenotypes resulting from the expression of plasmid-borne AtRRP44A, AtRRP44B/SOV, AtRRP44A and AtRRP44B/SOV, and ScRrp44 in <i>S. cerevisiae</i> BSY1883 strain, and negative control alleles were assessed in the presence (repressed chromosomal ScRrp44) or absence (expressed chromosomal ScRrp44) of DOX after incubation for 90 h at 30°C. –LEU-TRP, without leucine and tryptophan. (C) Diagram of the intron–exon structure of AtRRP44A and AtRRP44B/SOV. UTRs are indicated by grey boxes, exons by black boxes and introns by solid lines. T-DNA insertion sites for <i>rrp44a-1</i> (SALK_037533), <i>rrp44a-2</i> (SALK_141741), <i>rrp44a-3</i> (SALK_051800), <i>rrp44b-1</i> (SAIL_804_F05), <i>rrp44b-2</i> (SALK_017934) and <i>rrp44b-3</i> (SALK_010765) are shown in red arrowheads.</p

Levels of selected subsets of RNAs in <i>rrp44aKD-1</i>, <i>rrp44b-2</i> and the exosome core mutants.

<p>qRT-PCR analysis of total RNAs isolated from 7 dpg leaves for AtRRP4 and AtRRP41 (AT5G11090 3ʹ extension, AT5G27720-Intron) and AtRRP41L (NCED3) substrates [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0079219#B10" target="_blank">10</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0079219#B30" target="_blank">30</a>] (A–C). UTRs are indicated by grey boxes, exons by black boxes, introns by solid lines and the 3ʹ extended region by a black broken line (A and B). Green lines show the coverage of amplicons used for qRT-PCR. Error bars represent standard errors. Three biological replicates and two technical replicates were performed. EF1a mRNA was used as an endogenous control. * indicates significant difference (<i>p</i> < 0.05, Tukey’s test) between mutants and <i>gusKD-2</i> (VC) or Col-0 (WT) plants. </p

Establishment of <i>rrp44a</i> knock-down mutants by artificial microRNA (amiR).

<p>(A) Schematics of the amiR precursor. A black circle represents cap structure. amiR and amiR* represent guide strand and passenger strand, respectively. amiR sequences targeting AtRRP44A (amiR_AtRRP44A-1 and amiR_AtRRP44A-2) and <i>E. coli</i> β-glucuronidase (GUS) (amiR_GUS-2; vector control with no target sites in the <i>Arabidopsis</i> genome) are shown. (B) AmiR sequences and target sites in the AtRRP44A (AT2G17510) mRNA. (C) Expression of amiR_RRP44A-1 and amiR_RRP44A-2 were detected by small RNA gel blot analysis. Small RNA gel blots were hybridized with an antisense oligonucleotide complementary to the amiRs. U6 RNA (U6) served as a loading control for small RNA. Total RNAs were isolated from 25 days post-germination (dpg) rosette leaves of T3 homozygous lines carrying a unique insertion in Col-0 background plants expressing amiR_RRP44A-1 (AtRRP44A Knocked Down-1; <i>rrp44aKD-1#</i>7-3-1), amiR_RRP44A-2 (<i>rrp44aKD-2</i>#6-2-1), or amiR_GUS-2 (gusKD-2#2-10-3: vector control (VC)). (D) The amounts of AtRRP44A mRNA in <i>gusKD-2</i>#2-10-3, <i>rrp44aKD-1</i>#7-3-1 and <i>rrp44aKD-2</i>#6-2-1 were analyzed by qRT-PCR. Total RNAs were isolated from 25 dpg rosette leaves. Error bars represent standard errors. Six biological replicates and two technical replicates were performed. * indicates significant difference (<i>p</i> < 0.01, Tukey’s test) between <i>gusKD-2</i> (VC) and <i>rrp44aKD-1</i> and <i>-2</i>. </p

Levels of MRP RNA and snoRNA31 in <i>rrp44aKD-1</i>, <i>rrp44b-2</i> and the exosome core mutants.

<p>(A and B) qRT-PCR revealed that accumulation of the MRP RNA and snoRNA31 was upregulated in <i>rrp4KD-3</i>, <i>rrp41KD-1</i> and <i>rrp44aKD-1</i>, but not in <i>rrp44b-1</i>. AtRRP4 and AtRRP41 represent the <i>Arabidopsis</i> exosome core. Total RNAs were isolated from 10 dpg rosette leaves. EF1a mRNA was used as an endogenous control. Error bars represent standard errors. Three biological replicates and two technical replicates were performed. * indicates significant difference (<i>p</i> < 0.05, Tukey’s test) between mutant and wild type plants. </p

Timm’s staining in the hippocampus.

<p>Hippocampal slices were prepared from Wistar rats (A) and NER (B) (n = 10). The lower panel show the density of Timm’ stain, which was measured by using Multi Gauge V3.1, in the stratum lucidum where mossy fiber terminals exist. As a representative sample (circle) in Fig. 1A, five regions of interest per slice were set in the stratum lucidum and the densities measured were averaged. Each bar and line (mean ± SEM) represents the rate (%) of the density of Timm’ stain of Wistar rats to that of NER, which was represented as 100%.</p

Suppression of CQ-induced seizures in NER by enhancing GABAergic activity.

<p>AOAA (5, 10, 20 mg/kg) or phenobarbital (PHB, 20 mg/kg) was i.p. co-injected with CQ (30 mg/kg) into NER, and then the behavior was observed for 6 h in home cage. The incidence represents the rate of seized rats, which exhibited tonic-clonic convulsion, to the total rats (vehicle, n = 22; CQ 30 mg/kg, n = 38; CQ+AOAA (5 mg/kg), n = 10; CQ+AOAA (10 mg/kg), n = 10; CQ+AOAA (20 mg/kg), n = 20; CQ+PHB (20 mg/kg), n = 18). Each bar and line represents the mean ± SEM (A). ***, p<0.001, vs. vehicle; ^#, p<0.05, ^##, p<0.01, ^###, p<0.001, vs. CQ. To check the effect of AOAA on locomotor activity, NER were subjected to the open-field test 2 h after i.p. injection of AOAA (n = 10). Each bar and line represents the mean ± SEM (B). *, p<0.05, vs. vehicle.</p

Enhanced exocytosis in mossy fibers after CQ injection.

<p>Hippocampal slices (400- µm thickness) were prepared 2 h after i.p. injection of CQ (30 mg/kg). To measure the decrease in FM4-64 fluorescence intensity in mossy fiber terminals (boutons), mossy fiber terminals were doubly stained with ZnAF-2 and FM4-64. ZnAF-2 staining was done under an optimal condition to stain mossy fiber terminals clearly, unlike the ZnAF-2 staining condition of Fig. 4. The terminals strongly stained with ZnAF-2 were determined as region of interest. To observe presynaptic activity, tetanic stimulation at 10 Hz for 30 s, which was shown by a shaded bar, was delivered to mossy fibers and then single strong stimulation at 100 Hz for 18 s was delivered to the same position. The activity-dependent component of FM4-64 signal was measured for each punctum (1 s) by subtracting its residual fluorescence intensity measured by the strong electrical stimulation. The data (mean ± SEM) represent the ratio (%) of each FM4-64 intensity to the basal FM4-64 intensity before tetanic stimulation at 10 Hz for 30 s, which was averaged and expressed as 100% (vehicle, n = 10; CQ, n = 12). SL, stratum lucidum; PCL, CA3 pyramidal cell layer. *, p<0.05, vs. vehicle.</p