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: Biology of reproduction

Article Title: Pre-messenger RNA cleavage factor I (CFIm): potential role in alternative polyadenylation during spermatogenesis.

doi: 10.1095/biolreprod.107.064774

Figure Lengend Snippet: FIG. 1. A novel Nudt21 RNA is highly enriched in mouse testis and male germ cells. A) Schematic of male germ cell and somatic Nudt21 transcripts for mouse, showing that structural differences occur in their respective 30UTRs. DNA products generated by RT-PCR (A, B, C, E) and 3’RACE (D) from mouse testis or kidney total RNA are shown below the transcripts. Arrows indicate the position of the exon 1/intron 1 junction and translation initiation sites. B) Northern blot of total RNA isolated from mouse tissues and enriched adult male germ cells using RT-PCR product A as a probe (see A). Lanes: mouse testis (T), enriched adult male germ cells (gc), kidney (K), liver (L), brain (B), and heart (H). Ethidium bromide staining of RNA loading is shown below each lane. C) Northern analysis of mouse testis and kidney total RNA using a probe specific to Nudt21 exon 1 (product B, see A). Ethidium bromide staining of ribosomal RNAs is shown below. D) Hybridization of total RNA from mouse tissues with RT-PCR product E specific to the 30UTR of mouse somatic Nudt21 mRNA (see A). The blot used in the right panel of B was reprobed for somatic-specific 30UTR sequences and detected a ;4.5-kb mRNA in brain and other tissues, but not the 1.1-kb testis-enriched transcript.

Article Snippet: After blocking with 1% nonfat milk for 1 h at room temperature, the blots were incubated with antibody raised against NUDT21/ CPSF5 (1:1000; #BC001403; ProteinTech Group, Chicago, IL) or CPSF6 (1:1000; Abnova, Taipei City, Taiwan) overnight at 48C.

Techniques: Generated, Reverse Transcription Polymerase Chain Reaction, Northern Blot, Isolation, Staining, Hybridization

Journal: Biology of reproduction

Article Title: Pre-messenger RNA cleavage factor I (CFIm): potential role in alternative polyadenylation during spermatogenesis.

doi: 10.1095/biolreprod.107.064774

Figure Lengend Snippet: FIG. 2. Nucleotide and coding sequences for mouse testis Nudt21. A) Composite cDNA sequence for mouse testis Nudt21. Primers used to amplify various RT-PCR and 3’RACE products are underlined. The translational initiation and stop codons (bold) as well as putative canonical and noncanonical polyadenylation signals (dashed boxes) and upstream CFIm binding sites (solid line boxes) in the 30UTR are indicated. B) Predicted coding sequences for NUDT21 from mouse testis and somatic human NUDT21 isoforms (hNUDT21–1, 2). See text for GenBank Accession numbers. Asterisks indicate identity with the predicted mouse testis amino acid sequence, while residues absent in the human splice variant hNUDT21–2 are shown with dashes.

Article Snippet: After blocking with 1% nonfat milk for 1 h at room temperature, the blots were incubated with antibody raised against NUDT21/ CPSF5 (1:1000; #BC001403; ProteinTech Group, Chicago, IL) or CPSF6 (1:1000; Abnova, Taipei City, Taiwan) overnight at 48C.

Techniques: Sequencing, Reverse Transcription Polymerase Chain Reaction, Binding Assay, Variant Assay

Journal: Biology of reproduction

Article Title: Pre-messenger RNA cleavage factor I (CFIm): potential role in alternative polyadenylation during spermatogenesis.

doi: 10.1095/biolreprod.107.064774

Figure Lengend Snippet: FIG. 6. Distinct developmental variation of CFIm subunit mRNAs and proteins during male germ cell development. A) Total RNA from purified type A spermatogonia (lane A), type B spermatogonia (lane B), prepubertal pachytene spermatocytes (lane PP), pachytene spermatocytes (lane PS), round spermatids (lane RS), and residual bodies (RB) were hybridized with Nudt21 probe A and Cpsf6 probe F. The same blot was probed in each case. Ethidium bromide staining of RNA loading is shown below. B) NUDT21 and CPSF6 protein concentrations are similar in mitotic, meiotic, and spermiogenic germ cell populations. B, B type spermatogo- nia; PS, pachytene spermatocytes; RS, round spermatids; gc, enriched adult mouse male germ cells.

Article Snippet: After blocking with 1% nonfat milk for 1 h at room temperature, the blots were incubated with antibody raised against NUDT21/ CPSF5 (1:1000; #BC001403; ProteinTech Group, Chicago, IL) or CPSF6 (1:1000; Abnova, Taipei City, Taiwan) overnight at 48C.

Techniques: Purification, Staining

Journal: Biology of reproduction

Article Title: Pre-messenger RNA cleavage factor I (CFIm): potential role in alternative polyadenylation during spermatogenesis.

doi: 10.1095/biolreprod.107.064774

Figure Lengend Snippet: FIG. 5. CFIm subunit proteins are elevated in mouse testis and male germ cells. A) Western analysis of NUDT21 (left panel) and CPSF6 (right panel) proteins in whole testis (T) and kidney (K). Equivalency of protein loading was confirmed by Ponceau-S staining (not shown). B) NUDT21 (left) and CPSF6 (right) proteins in nuclear extracts of enriched adult mouse spermatogenic cells (gc).

Article Snippet: After blocking with 1% nonfat milk for 1 h at room temperature, the blots were incubated with antibody raised against NUDT21/ CPSF5 (1:1000; #BC001403; ProteinTech Group, Chicago, IL) or CPSF6 (1:1000; Abnova, Taipei City, Taiwan) overnight at 48C.

Techniques: Western Blot, Staining

Journal: Biology of reproduction

Article Title: Pre-messenger RNA cleavage factor I (CFIm): potential role in alternative polyadenylation during spermatogenesis.

doi: 10.1095/biolreprod.107.064774

Figure Lengend Snippet: FIG. 8. CFIm transcripts are enriched in human testis. Northern blots of total RNA from different human tissues were probed for Nudt21 (upper panel; probe A) and Cpsf6 (lower panel; probe F) mRNAs. Ethidium bromide staining is shown below for each blot. Lanes: human liver (L), pancreas (P), heart (H), lung (Lu), muscle (M), kidney (K), spleen (S), testis (T), and small intestine (SI).

Article Snippet: After blocking with 1% nonfat milk for 1 h at room temperature, the blots were incubated with antibody raised against NUDT21/ CPSF5 (1:1000; #BC001403; ProteinTech Group, Chicago, IL) or CPSF6 (1:1000; Abnova, Taipei City, Taiwan) overnight at 48C.

Techniques: Northern Blot, Staining

Journal: Biology of reproduction

Article Title: Pre-messenger RNA cleavage factor I (CFIm): potential role in alternative polyadenylation during spermatogenesis.

doi: 10.1095/biolreprod.107.064774

Figure Lengend Snippet: FIG. 9. ChIP analysis of NUDT21 in adult mouse germ cells and kidney. Formalde- hyde-cross-linked chromatin was precipi- tated with antibodies to NUDT21 (Anti- NUDT21) or using beads alone without primary antibody (No Ab Ctl). A) Following reversal of crosslinks, DNA precipitated from adult male germ cells was assayed for mouse Srebf2, Nr6a1, and Gabra6 genomic sequences by PCR. Specific bands generat- ed by the respective PCR reactions are shown in each case. Aliquots of input DNA (Input) were assayed as positive controls. B) NUDT21 ChIP analysis of proximal (Srebf2_v1) and distal (Srebf2) 30UTRs for the mouse Srebf2 gene in mouse sper- matogenic cell (Germ cells) and kidney chromatin.

Article Snippet: After blocking with 1% nonfat milk for 1 h at room temperature, the blots were incubated with antibody raised against NUDT21/ CPSF5 (1:1000; #BC001403; ProteinTech Group, Chicago, IL) or CPSF6 (1:1000; Abnova, Taipei City, Taiwan) overnight at 48C.

Techniques: Genomic Sequencing