Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Genome-wide CRISPR screen identifies HNRNPL as a prostate cancer dependency regulating RNA splicing

doi: 10.1073/pnas.1617467114

Figure Lengend Snippet: HNRNPL directly regulates alternative splicing in prostate cancer. (A) Pie chart showing different classes of HNRNPL-dependent alternative splicing events. (B) HNRNPL binding is significantly associated with HNRNPL-dependent alternative splicing. Fraction of genes with HNRNPL binding from differential splicing genes was compared with that from all genes. (C) Genome browser representation of HNRNPL binding over AR loci from RIP-seq data. (D) RT-qPCR confirmation of HNRNPL-dependent alternative splicing of AR. Both two alternatively spliced isoforms and pre-mRNA of AR were examined using corresponding PCR primers by RT-qPCR analysis upon HNRNPL knockdown. Data were shown as mean ± SEM, n = 3, *P < 0.05. (E) RIP-qPCR confirmation of HNRNPL binding on AR RNA. Data were shown as mean ± SEM, n = 3. (F) Western blot analysis of full-length AR protein level upon HNRNPL knockdown. GAPDH served as loading control.

Article Snippet: The antibodies were purchased from the following sources: GAPDH (FL-335, Santa Cruz, Cat. no. sc-25778), HNRNPL (4D11, Santa Cruz, Cat. no. sc-32317; D5, Santa Cruz, Cat. no. sc-48391), HNRNPK (3C2, Santa Cruz, Cat. no. sc-32307), and AR (N-20, Santa Cruz, Cat. no. sc-816).

Techniques: Alternative Splicing, Binding Assay, Quantitative RT-PCR, Knockdown, Western Blot, Control

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Genome-wide CRISPR screen identifies HNRNPL as a prostate cancer dependency regulating RNA splicing

doi: 10.1073/pnas.1617467114

Figure Lengend Snippet: HNRNPL is involved in CircRNA regulation. (A) Schematic depiction of circRNA formation via back splicing of linearly aligned exons. (B) Correlation of fold-change in abundance of HNRNPL-regulated circRNA (y axis) and its cognate parental mRNA (x axis). Note the scale difference between the two axes. (C) Association of HNRNPL binding status with circRNA expression regulation. The fold-enrichment represents the fraction for each category over all circRNA genes. (*P < 0.05). (D) RT-qPCR analysis of several HNRNPL-regulated circRNAs and their cognate pre-mRNAs. Validated circRNAs are selected as more than ∼twofold change upon HNRNPL knockdown and with HNRNPL binding in any region of parental genes from Dataset S4. Data are shown as mean ± SEM, n = 3, *P < 0.05. (E) Genome browser representation of HNRNPL binding over PPFIA2 loci as determined by RIP-seq. The back-spliced exons that generate two different circRNAs (circ-1 and circ-2) are denoted. The two flanking HNRNPL-binding peaks are highlighted. (F) RIP-qPCR validation of two HNRNPL-binding peaks over PPFIA2 transcript highlighted in E. Data are shown as mean ± SEM, n = 3. (G) Sanger sequencing traces for confirmation of back-spliced exon junctions of the two separate PPFIA2 circRNA. (H) Schematic description showing the design of GAPDH minigene assay. (I) RT-qPCR analysis of relative circRNA formation normalized by pre-mRNA levels for different GAPDH minigenes transfected into HEK293T cells. Data are shown as mean ± SEM, n = 3. (J) CircRNA formation of the GAPDH minigene is dependent on HNRNPL. RT-qPCR analysis was performed in G4 minigene-transfected samples upon control or HNRNPL knockdown. Data are shown as mean ± SEM, n = 3.

Article Snippet: The antibodies were purchased from the following sources: GAPDH (FL-335, Santa Cruz, Cat. no. sc-25778), HNRNPL (4D11, Santa Cruz, Cat. no. sc-32317; D5, Santa Cruz, Cat. no. sc-48391), HNRNPK (3C2, Santa Cruz, Cat. no. sc-32307), and AR (N-20, Santa Cruz, Cat. no. sc-816).

Techniques: Binding Assay, Expressing, Quantitative RT-PCR, Knockdown, Biomarker Discovery, Sequencing, Mini Gene Assay, Transfection, Control

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Genome-wide CRISPR screen identifies HNRNPL as a prostate cancer dependency regulating RNA splicing

doi: 10.1073/pnas.1617467114

Figure Lengend Snippet: CircRNA Regulation by HNRNPL. (A) RT-qPCR analysis of indicated circRNAs and mRNAs before and after RNase R treatment. (B) Sanger sequencing traces for confirmation of back-spliced exon junctions of indicated circRNAs. (C) Sanger sequencing traces for confirmation of back-spliced exon junction of the GAPDH circRNA from minigene assay. (D) Bar plot showing the relationship between the fractions of genes that form circular transcripts and the number of HNRNPL binding sites that they have.

Article Snippet: The antibodies were purchased from the following sources: GAPDH (FL-335, Santa Cruz, Cat. no. sc-25778), HNRNPL (4D11, Santa Cruz, Cat. no. sc-32317; D5, Santa Cruz, Cat. no. sc-48391), HNRNPK (3C2, Santa Cruz, Cat. no. sc-32307), and AR (N-20, Santa Cruz, Cat. no. sc-816).

Techniques: Quantitative RT-PCR, Sequencing, Mini Gene Assay, Binding Assay