PTB and <i>let-7</i> miRNA contribute together to regulate gene expression in <i>C. elegans</i>.

<p>(A) Synchronized L1 animals were placed at semi-permissive temperature (20°C) and adult animals were scored after seventieth-two hours. The animal sterility observed in the population is caused by either a vulval bursting at the L4-adult transition or by a severe gonadal defect. Error bars represent the 95% confidence interval from independent experiments (n) where between 20 and 40 animals have been scored. ***: p<0.0001 (B) <i>let-7</i> level remained unchanged in the <i>let-7ts</i>/<i>ptb-1</i> animals. RNAs were purified from the indicated genotypes and probed for <i>let-7</i> and U6 RNAs. The amount of RNA was used for Northern blotting is indicated on the top of the panel and the U6/<i>let-7</i> ratios are presented at the bottom of the panel.</p

Affinity purification of <i>let-7</i> associated complexes.

<p>(A) Biotinylated 2-<i>O</i>-methylated oligos used in this study. Sequences highlighted with red are complementary to <i>let-7a</i>. Blue nucleotides indicate changes generated from the original <i>let-7</i> oligo. (B) Northern hybridization (top panel) and Western blot (bottom panel) show that <i>let-7</i> oligo specifically purifies <i>let-7</i> miRNA and hAgo2 protein. sup.: supernatant; c and cont.: control oligo. (C) Proteins co-purify with <i>let-7</i> oligo. Right and left panels show the results of the independent affinity purifications. Proteins that are specifically pulled down with the <i>let-7</i> oligo are labeled next to the stained gels.</p

PTB association with the <i>let-7</i> bead depends of the <i>let-7</i> seed complementary sequences.

<p>(A) <i>let-7</i> seed mutant oligo could not inhibit <i>let-7</i> mediated gene repression. Renilla luciferase expressing plasmid containing a part of the 3′ UTR of human HMGA2 that carries four <i>let-7</i> target sites were transfected into HeLa cells together with Firefly expressing plasmid, as internal control, and the indicated 2′-<i>O</i>-methyl oligos. The graph shows the result of the dual-luciferase assay normalized to the control oligo. The error bars represent the standard error of three experiments. (B) <i>let-7</i>, hAgo2 and PTB are sensitive to the presence of the seed sequence of the <i>let-7</i> oligo. The quantity of <i>let-7</i> miRNA associated with the indicated oligos was quantified using Northern hybridization and normalized to the amount of miRNA pulled down with the wild-type <i>let-7</i> oligo. The presences of hAgo2 and PTB on the indicated beads were monitored by Western hybridization. (C) PTB association with the <i>let-7</i> column does not depend on the presence of the canonical PTB site in the oligo. Affinity purifications were carried out with the indicated oligos and the association of miRNAs, hAgo2 and PTB with these oligos was monitored by Northern hybridization and Western blotting. sup.: supernatant.</p

PTB is associated with hAgo2 and <i>let-7</i> miRNA.

<p>Endogenous PTB in Hela cells (A), PTB fused with GFP in HeLa cells (B) and stably expressed GFP::PTB in U2OS cells (C) co-purify with endogenous hAgo2 and <i>let-7</i>. Immunoprecipitations (IP) were carried out with the indicated antibodies. The bound fractions were assayed for hAgo2 and PTB with western blotting (top panels) and for <i>let-7</i> with Northern hybridization (bottom panels). (D) PTB association with Ago2 is mediated by RNA. IPs were carried out with antibodies against GFP and PTB. The parts of the bound fraction were subjected to RNase treatment and the supernatants of the RNAse treated beads and the remaining bound fractions were assayed for hAgo2 and PTB by Western blotting.</p

nPTB could also be associated with miRISC.

<p>(A) The knock down of PTB results in the increase of nPTB expression in HeLa cells. PTB was knock down with specific siRNA and PTB and nPTB levels were monitored with Western blotting. Tubulin was used as a loading control. *: non-specific hybridization visualized by he nPTB abtibody. (B) nPTB is associated with miRNA. XR tagged nPTB was overexpressed and IP was carried out with antibody recognizing XR. The efficiency of the IP was checked with Western blotting using XR and nPTB antibodies. RNA was purified from the immunoprecipitates and assayed for the presence of <i>let-7</i> using Northern blotting. c: empty bead.</p

PTB alters Ago2 association of mRNAs in HeLa cells.

<p>(A) PTB and nPTB was simultaneously knocked down in triplicates in HeLa cells and Ago2 was immunoprecipitated from control and PTB/nPTB siRNA transfected cells. PTB, nPTB, Ago2 expression was followed by Western hybridization. Tubulin was used as loading control. *: non-specific band detected with the nPTB antibody. (B–F) q-PCR analysis of mRNAs which association with Ago2 is modulated by PTB. RNAs were isolated from control and PTB/nPTB siRNA transfected cells and from Ago2 IPs obtained from the same cells. RNAs were quantified and normalized with GAPDH RNA. The data show the relative abundance of the normalized RNAs compared to the control siRNA transfected cells and the Ago2 IP from the same cells. Error bars represent the standard deviation of three independent experiments (A). *: p<0.05, **: p<0.001.</p

Deletion of miR212/132 influences hippocampal synaptic transmission and plasticity.

<p>A) Plot of fEPSP slope vs stimulus strength (mean ± SEM) showing that basal synaptic transmission is reduced by ∼30% in miR 212/132 KO slices (grey circles) compared to control (fl/fl) slices (black circles). Inset are representative fEPSPs at each of the stimulus strengths for fl/fl (left, black traces) and KO (right, grey traces) slices. Scale bar measures 5 ms and 1 mV. B) Paired-pulse facilitation (mean ± SEM) is no different between control (black circles) and miR KO slices (grey circles). Inset are representative fEPSPs at each of the inter-pulse intervals for control (left, black traces) and KO (right, grey traces) slices. Scale bar measures 100 ms and 1 mV. A and B represent data from 17 slices from 8 KO, and 11 slices from 6 control mice. C) LTP induced by tetanic stimulation (100 Hz for 1s) was not appreciably different between fl/fl (black circles) and KO mice (grey circles). Inset are fEPSPs taken before (smaller of the traces) and 60 mins after the induction of LTP (larger of the two traces) in fl/fl (left, black traces) and KO (right, grey traces) slices. Data from 11 slices from 8 KO mice and 6 slices from 4 control mice. Mean ± SEM. Scale bar measures 5 ms and 1 mV. D) In contrast, theta-burst stimulation (5 episodes at 10 s intervals of theta-burst stimulation (5 pulses at 100 Hz, repeated 10 times with 200 ms interval)) revealed greater LTP in KO slices (grey circles) compared to fl/fl (black circles) slices (p<0.05 at 55–65^th minute; unpaired t-test). Inset are fEPSPs taken before (smaller of the traces) and 60 mins after the induction of LTP (larger of the two traces) in fl/fl (left, black traces) and KO (right, grey traces) slices. The black and grey triangles hovering at 100% refer to the slope of fEPSPs from a simultaneously recorded control pathway for fl/fl and KO slices, respectively. Data from 13 slices from 5 KO mice and 6 slices from 2 fl/fl mice. Scale bar measures 5 ms and 1 mV. Data is presented as mean ± SD.</p

Generation of miR-132/212 knockout mice.

<p>miR-132/212 knockout mice were generated by insertion of LoxP sites in the 1^st intron and exon2 of the small non coding RNA gene that contains miR-132 and miR-212 (A). Targeted C57BL/6 ES cells were generated as described in the Methods section, and identified by Southern analysis of Kpn I digests using a probe 3′ to the sequence used in the targeting vector (B). Correctly targeted ES cells were used to generate a conditional miR-132/212 allele in mice using standard techniques. Mice were crossed to Flp transgenic mice to excise Neomycin resistance cassette and then Cre expressing mice to delete miR-132 and miR-212. Routine genotyping was carried out using 3 primers (p1, p2 and p3) which resulted in bands of 373 bp for the wild-type allele, 420 for the floxed allele and 550 for the deleted allele (C). Litter sizes (D) and pre weaning mortality (E) for matings of male and female knockout (n = 50 for litter size, n = 39 for pre weaning mortality), floxed (n = 20 or 17) or heterozygous (n = 53 or 41) mice are shown. Litters where pups were used for neuronal cultures were excluded from the pre weaning mortality values. Error bars represent standard deviation.</p

Primer sequences used for qPCR.

<p>Primer sequences used for qPCR.</p

miR-132 and miR-212 are not required for cytokine induction in BMDMs.

<p>BMDMs were isolated from wild-type or miR-132/212 knockout mice. Cells were either left unstimulated or stimulated with 100 ng/ml LPS, 10 µg/ml poly I:C, 2 mM CpG, 100 ng/ml Pam3-CSK4, 100 ng/ml Pam3-CSK4 or 100 ng/ml CL097 for 6 h. Secreted levels of TNF, IL-12p40, IL-6 and IL-10 in the media were measured by a multiplex based assay as described in the methods. Error bars represent the standard deviation of independent cultures from 4 mice per genotype.</p