Journal: eLife

Article Title: Partial loss of CFIm25 causes learning deficits and aberrant neuronal alternative polyadenylation

doi: 10.7554/eLife.50895

Figure Lengend Snippet: ( A ) Immunofluorescence showing CFIm25 expression in the nuclei of some cells in the (i) mouse hippocampus CA1 region and (ii) cortex. Arrows indicate nuclei that do not have CFIm25. ( B ) Immunofluorescence of the mouse cortex showing colocalization of CFIm25 and the neuronal marker NeuN. Arrows indicate a nucleus with CFIm25 and NeuN expression. ( C ) Schematic of the floxed and recombined Nudt21 alleles (top), and sequencing showing successful recombination (bottom). ( D ) Observed and expected offspring counts born with each possible genotype from Nudt21 +/- mating pairs. No homozygous Nudt21 null offspring were born, when eight would be expected if loss of Nudt21 did not affect survival: p=0.004, analyzed by two-tailed, chi-square test. ( E ) Schematic of wild-type and recombined Nudt21 mRNA (top). Triangles indicate RT-qPCR primer binding sites, and UAA shows site of induced premature stop codon after recombination. RT-qPCR analysis shows expected 50% reduction of whole-brain, Gapdh -normalized, wild-type Nudt21 mRNA in five-week-old mice with one wild-type Nudt21 allele and one recombined, null allele (bottom): p<0.0001, n = 3–5/genotype. ( F ) Western blot image comparing five-week-old Nudt21 +/- mice CFIm25 protein levels with their WT littermates (top). Western blot analysis showing ~30% reduction of H3-normalized CFIm25 protein levels in Nudt21 +/- mice: p=0.0012, n = 3–5/genotype. We confirmed that CFIm25 does not regulate H3. For all charts, error bars indicate SEM. All data analyzed by unpaired, two-tailed t-test unless otherwise stated. **p<0.01; ****p<0.0001. Weights of the heterozygous animals are shown in .

Article Snippet: We then incubated the sections in siRNA-validated CFIm25 primary antibody (1:50, Proteintech 10322–1-AP) in blocking buffer overnight at 4°C, followed by three washes and Alexa Fluor 488 (1:1000, Molecular Probes) secondary antibody incubation for two hours at room temperature or anti-NeuN primary antibody (1:50, Millipore MAB377) and Alexa Fluor 555 (1:1000).

Techniques: Immunofluorescence, Expressing, Marker, Sequencing, Two Tailed Test, Quantitative RT-PCR, Binding Assay, Western Blot

Journal: eLife

Article Title: Partial loss of CFIm25 causes learning deficits and aberrant neuronal alternative polyadenylation

doi: 10.7554/eLife.50895

Figure Lengend Snippet: ( A ) Schematic of NUDT21 mRNA with qPCR primers (top) and RT-qPCR quantification of NUDT21 mRNA levels in human embryonic stem cell (ESC)-derived neurons infected with scrambled shRNA (shScramble) or shRNA targeting NUDT21 (sh NUDT21 ). The neurons infected with sh NUDT21 have a 40% reduction of GAPDH -normalized NUDT21 : p=0.0009, n = 3–4/treatment. ( B ) Western blot image and quantification showing a 30% reduction of H3-normalized CFIm25 protein in sh NUDT21 -infected neurons: p=0.0005, n = 4/treatment. We confirmed that CFIm25 does not regulate H3. ***p<0.001.

Article Snippet: We then incubated the sections in siRNA-validated CFIm25 primary antibody (1:50, Proteintech 10322–1-AP) in blocking buffer overnight at 4°C, followed by three washes and Alexa Fluor 488 (1:1000, Molecular Probes) secondary antibody incubation for two hours at room temperature or anti-NeuN primary antibody (1:50, Millipore MAB377) and Alexa Fluor 555 (1:1000).

Techniques: Quantitative RT-PCR, Derivative Assay, Infection, shRNA, Western Blot

Journal: eLife

Article Title: Partial loss of CFIm25 causes learning deficits and aberrant neuronal alternative polyadenylation

doi: 10.7554/eLife.50895

Figure Lengend Snippet: ( A )(i) Poly(A)-ClickSeq (PAC-seq) track showing reduced NUDT21 and (ii-iv) PAC-seq tracks showing altered alternative polyadenylation in example genes MECP2 , VMA21 , and PAK1 . n = 3/treatment. Peaks are 3′ end sequencing reads. Multiple peaks in a gene indicate multiple mRNA isoforms with different cleavage and polyadenylation sites. Bracketed numbers show counts per million; kb stands for kilobases. ( B ) Schemata of the mRNA isoforms identified by PAC-seq for MECP2 , VMA21 , and PAK1 (top). Triangles show the binding sites of the qPCR primers used to validate the PAC-seq results. The gray primers detect all mRNA isoforms for the target gene, whereas the green primers only detect the isoform with the longest 3′ UTR. Gapdh -normalized RT-qPCR quantification showing relatively less of the long isoforms in the sh NUDT21 -infected neurons (bottom) for MECP2 , p=0.0007 (i), VMA21 , p=0.01 (ii), and PAK1 , p=0.005 (iii). Error bars indicate SEM. Data analyzed by unpaired, two-tailed t-test. ( C ) Volcano plot showing relative mRNA length change in sh NUDT21 -infected neurons compared to shScramble-infected controls. The horizontal, dashed line shows P adjusted = 0.05, n = 3/treatment. NUDT21 loss in neurons predominantly results in shorter mRNAs (p<0.0001, two-tailed chi-square test). >90% of reads are in 3′ UTRs . Principle component analysis (PCA) shows sample separation by treatment . Distal cleavage sites of NUDT21 -regulated mRNAs are enriched for the CFIm25 binding motif, UGUA, in mRNAs that shorten after NUDT21 knockdown, but not in non-target mRNAs . ( E ) Mass spectrometry quantification of protein level fold change for genes with significantly altered APA (P adjusted <0.05). mRNA shortening predominantly results in increased protein levels (p<0.0001, two-tailed conditional chi-square test). Source files for the PAC-seq and mass spectrometry quantification data are available in . *p<0.05; **p<0.01; ***p<0.001. Figure 6—source data 1. RNA length and protein level changes in genes with misregulated APA following NUDT21 inhibition in human neurons. Alternative polyadenylation (APA), likelihood ratio (lr), degrees of freedom (df), first polyadenylation site (F), middle/center polyadenylation site (M), last polyadenylation site (L) mass spectrometry (MS), fold change (FC), intellectual disability (ID), probability of loss of function intolerance (pLI).

Article Snippet: We then incubated the sections in siRNA-validated CFIm25 primary antibody (1:50, Proteintech 10322–1-AP) in blocking buffer overnight at 4°C, followed by three washes and Alexa Fluor 488 (1:1000, Molecular Probes) secondary antibody incubation for two hours at room temperature or anti-NeuN primary antibody (1:50, Millipore MAB377) and Alexa Fluor 555 (1:1000).

Techniques: Sequencing, Binding Assay, Quantitative RT-PCR, Infection, Two Tailed Test, Mass Spectrometry, Inhibition

Journal: eLife

Article Title: Partial loss of CFIm25 causes learning deficits and aberrant neuronal alternative polyadenylation

doi: 10.7554/eLife.50895

Figure Lengend Snippet: ( A ) >90% of filtered reads map to the 3′ UTR. ( B ) Control and sh NUDT21 -infected human neurons segregate by principle component analysis (PCA) of alternative polyadenylation. (Ci) The CFIm25 binding motif, UGUA, is enriched upstream of the distal cleavage and polyadenylation site (PAS) of NUDT21 -regulated mRNAs that shorten after NUDT21 knockdown. (ii) There is almost no upstream UGUA frequency difference between proximal and distal cleavage sites in mRNAs not affected by NUDT21 loss. (iii) There is a slight UGUA enrichment upstream of the proximal cleavage site in NUDT21 -regulated mRNAs that lengthen after NUDT21 loss.

Article Snippet: We then incubated the sections in siRNA-validated CFIm25 primary antibody (1:50, Proteintech 10322–1-AP) in blocking buffer overnight at 4°C, followed by three washes and Alexa Fluor 488 (1:1000, Molecular Probes) secondary antibody incubation for two hours at room temperature or anti-NeuN primary antibody (1:50, Millipore MAB377) and Alexa Fluor 555 (1:1000).

Techniques: Infection, Binding Assay

Journal: eLife

Article Title: Partial loss of CFIm25 causes learning deficits and aberrant neuronal alternative polyadenylation

doi: 10.7554/eLife.50895

Figure Lengend Snippet:

Article Snippet: We then incubated the sections in siRNA-validated CFIm25 primary antibody (1:50, Proteintech 10322–1-AP) in blocking buffer overnight at 4°C, followed by three washes and Alexa Fluor 488 (1:1000, Molecular Probes) secondary antibody incubation for two hours at room temperature or anti-NeuN primary antibody (1:50, Millipore MAB377) and Alexa Fluor 555 (1:1000).

Techniques: Recombinant, shRNA, Sequencing, Amplification, Software, Mass Spectrometry

Journal: RNA Biology

Article Title: WDR33 alternative polyadenylation is dependent on stochastic poly(a) site usage and splicing efficiencies

doi: 10.1080/15476286.2024.2408708

Figure Lengend Snippet: CFIm25 variably regulates V2 and V3 PAS usage.

Article Snippet: Primary antibodies were: rabbit-anti-NUDT21 (CFIm25) (ABclonal, A4482); rabbit-anti-FIP1L1 (FIP1) (ABclonal, A7138), rabbit-anti-CstF64 [ ]; rabbit anti-Actin (Sigma-Aldrich, A2066) and rabbit anti-GAPDH (Sigma-Aldrich, G9545).

Techniques:

Journal: Nucleic Acids Research

Article Title: Knock-down of 25 kDa subunit of cleavage factor Im in Hela cells alters alternative polyadenylation within 3′-UTRs

doi: 10.1093/nar/gkl794

Figure Lengend Snippet: ( A ) A rabbit polyclonal antibody was raised against GST-CFIm25. A glutathione S -transferase (GST)-fusion protein of CFIm25 was expressed in E.coli and purified by affinity chromatography on glutathione Sepharose (GE Healthcare). HeLa whole cell extracts (20 μg) were separated on 12% SDS–polyacrylamide gels and analyzed by western blotting with pre-immune serum (lane 1) or anti-serum (lane 2). The arrowhead at right indicates CFIm25. ( B ) Knock-down of CFIm25. Two siRNAs (siRNA-408 and siRNA-613) were designed to target CFIm25. Whole cell extracts (20 μg) from mock-transfected (lane 1), siRNA transfected (control siRNA for lane 2, siRNA-408 for lane 3, siRNA-613 for lane 4) HeLa cells were subjected to western blot analysis with α-CFIm25, α-CstF-64 and α-actin antibodies. Actin was served as a loading control.

Article Snippet: The entire ORF of CF1m25 was cloned into the expression vector (pGEX-6P-1) and GST-CFIm25 protein was expressed in Escherichia coli and purified by affinity chromatography on glutathione Sepharose (GE healthcare). α-CFIm25 rabbit polyclonal antibody was raised against recombinant GST-CFIm25. α-CstF64 (H-300) was purchased from Santa Cruz and anti-actin (C4) was from Chemicon.

Techniques: Purification, Affinity Chromatography, Western Blot, Transfection