PRL1 is required for the accumulation of miRNAs.

<p>(A) The effect of various MAC components on the abundance of miRNAs (B) The levels of miRNAs are reduced in <i>prl1-2</i>. (C) The levels of siRNAs are reduced in <i>prl1-2</i>. Col: wild-type control. For miR159/319: upper band, miR159; lower band, miR319. Northern blot was used to detect small RNAs and the radioactive signals were quantified with ImageQuant (V5.2). To determine the amount of miRNAs/siRNAs in various mutants relative to that in Col, the radioactive signals of miRNAs/siRNAs were normalized to U6 RNA and compared with that in Col (set as 1). The numbers indicate the average value of three repeats (P<0.05).</p

PRL1 positively influences pri-miRNA levels.

<p>(A) The abundance of pri-miRNAs in inflorescences of <i>prl1-2</i> and Col. The transcript levels of pri-miRNAs in <i>prl1-2</i> were determined by quantitative RT-PCR (qRT-PCR), normalized to that of <i>UBQUITIN5</i> (<i>UBQ5</i>) and compared with those in Col. Value of Col was set to 1. Error bars indicate standard deviation of three technical replications. **: P<0.01. (B) GUS expression in <i>PRL1⁺</i> and <i>prl1-2</i> harboring <i>pMIR167a::GUS</i>. <i>PRL1⁺</i>: <i>PRL1/PRL1</i> or <i>PRL1/prl1-2</i>. Twenty plants containing GUS were stained for each genotype and an image was shown. (C) Relative <i>GUS</i> mRNA levels in <i>PRL1⁺</i> and <i>prl1-2</i> harboring <i>pMIR167a</i>::GUS. <i>GUS</i> transcript levels were determined by qRT-PCR and normalized to <i>UBQ5</i>. Value of <i>PRL1⁺</i> was set to 1. Standard deviation of three technical replications was shown as error bars. P = 0.11 (t-test).</p

The role of PRL1 in siRNA biogenesis.

<p>(A) PRL1 interacts with DCL3 and DCL4. Co-IP was performed to detect the interaction of PRL1 with DCL3 or DCL4. MBP and MBP-PRL1 fused protein were expressed in <i>E.coli</i>. YFP, DCL3-YFP and DCL4-YFP were expressed in <i>N. benthamiana</i> leaves. Anti-YFP was used for IP. For loading, ten percent and one percent of input proteins were used for IP and Co-IP, respectively. (B) <i>prl1-2</i> impairs siRNA production from double-stranded RNAs (dsRNAs). Protein extracts isolated from inflorescences of Col, <i>prl1-2</i> and <i>prl1-2</i> containing a PRL1-YFP transgene were incubated dsRNAs for 120 min. dsRNAs were synthesized through <i>in vitro</i> transcription of a DNA fragment (5′ portion of <i>UBQ5</i> gene, ∼460 bp) under the presence of [α-³²P] UTP.</p

PRL1 and CDC5 synergistically regulate miRNA accumulation.

<p>(A) Morphological phenotypes of Col, <i>cdc5-1</i>, <i>prl1-2</i> and <i>cdc5-1 prl1-2</i>. (B) The abundance of miRNAs is lower in <i>cdc5-1 prl1-2</i> than that in <i>cdc5-1</i> or <i>prl1-2</i>. Small RNAs were detected by Northern Blot. To determine the amount of miRNAs, radioactive signals of miRNAs were normalized to U6 RNA. The number represents the relative abundance compared to Col (set as 1) quantified by three repeats (P<0.05). (C) The abundance of pri-miRNAs is reduced in <i>cdc5-1 prl1-2</i>. The levels of pri-miRNAs in various mutants were determined by qRT-PCR, normalized to <i>UBQUITIN5</i> (<i>UBQ5</i>) and compared with those of Col (set as 1). Standard deviation of three technical replications was shown as error bars. **: P<0.01. (D) miR162b production from <i>pre-miR162b</i> in Col, <i>cdc5-1 prl1-2</i>, <i>cdc5-1</i> and <i>prl1-2</i>. The reaction was stopped at 120 min. The radioactive signals of miR162b were normalized to input. The number represents the relative production in various genotypes compared to Col (set as 1) quantified by three repeats (P<0.05).</p

PRL1 binds pri-miRNAs <i>in vitro</i> and <i>in vivo</i>.

<p>(A) Proteins used for <i>in vitro</i> RNA binding assay. Purified MBP and MBP-PRL1 proteins were resolved on SDS-page gel and stained by Coomassie Blue. The lower bands in MBP-PRL1 line are degraded MBP-PRL1 since they can be detected by anti-MBP antibody. M: marker. (B) PRL1 binds <i>MIR162b</i>, <i>pre-miR162b</i> and ssRNA <i>in vitro</i>. <i>MIR162b</i>, <i>pre-miR162b</i> and ssRNA were produced by <i>in vitro</i> transcription. SsRNA: single stranded-RNA; DsRNA: double stranded-RNA. (C) PRL1 binds pri-miRNAs <i>in vivo</i>. NoAb: No antibody control. Transgenic plants harboring <i>pPRL1::PRL1-YFP</i>, <i>pTGH:</i> TGH-YFP or <i>YFP</i> transgene were used for RIP assay with anti-YFP anti-bodies. Ten percent immunoprecipitates and two percent input proteins were analyzed by western blot. Five percent RNAs were used as input RNA.</p

PRL1 is required for miRNA maturation <i>in vitro</i>.

<p>(A) and (B) A schematic diagram of the <i>MIR162b</i> (A) and <i>pre-miR162b</i> (B) used <i>in vitro</i> processing assay. (C) and (D) The amount of miR162b produced from <i>MIR162b</i> and <i>pre-miR162b</i> were reduced in <i>prl1-2</i>. Proteins were isolated from inflorescences of <i>prl1-2</i> and Col and incubated with <i>MIR162b</i> or <i>pre-miR162b</i>. The reactions were stopped at various time points as indicated in the picture. (E) and (F) Quantification of miR162b production in <i>prl1-2</i> compared to that in Col. Quantification analysis was performed at 80 min. The radioactive signal of miR162 were normalized to input and compared with that of Col. The amount of miR162 produced in Col was set as 1. The value represents mean of three repeats (*** <i>P</i><0.001; t-test).</p

PRL1 associates with the Pol II and DCL1 complexes.

<p>(A) and (B) Co-immunoprecipitation (Co-IP) between PRL1 and Pol II. Protein extracts from transgenic plants containing PRL1-YFP were incubated with Anti-YFP or anti-RPB2 antibodies to precipitate PRL1-YFP or Pol II. PRL1-YFP and RBP2 were detected with western blot and labeled on the left side of the picture. Ten percent of input proteins were used for IP and one percent of input proteins were used for Co-IP. (C) BiFC analysis of PRL1 with DCL1, HYL1, SE, AGO1 and CDC5. Paired cCFP- and nVenus-fusion proteins were co-infiltrated into <i>N. benthamiana</i> leaves. The BiFC signal (Yellow fluorescence) was detected at 48 h after infiltration by confocal microscopy, assigned as green color and marked with arrow. 30 nuclei were examined for each pair and an image is shown. Red: auto fluorescence of chlorophyll. (D) and (E) Co-immunoprecipitation between PRL1 and DCL1. (F) and (G) Co-immunoprecipitation between PRL1 and SE. PRL1-YFP or YFP were co-expressed with DCL1-MYC and SE-MYC in <i>N. benthamiana</i>, respectively. Anti-YFP and anti-MYC (MBL) antibodies were used to detect YFP- and MYC-fused proteins, respectively. The protein pairs in the protein extracts were indicated on the on tope of the picture and proteins detected by western blot were indicated on the left side of the picture. Ten percent of input proteins were used for IP and one percent of inputs proteins were used for Co-IP.</p

Comparison between the results obtained by TCEA and the real objective values in the case of 20 dimensions.

Comparison between the results obtained by TCEA and the real objective values in the case of 20 dimensions.</p

Success rates of QBCA-2, BLCMAES, BLEAQ-2 and TCEA on 5, 10 and 20-dimensional test problems.

Success rates of QBCA-2, BLCMAES, BLEAQ-2 and TCEA on 5, 10 and 20-dimensional test problems.</p

Histogram of the median values on 5-dimensional problems.

Histogram of the median values on 5-dimensional problems.</p