Journal:

Article Title: Diverse roles of hnRNP L in mammalian mRNA processing: A combined microarray and RNAi analysis

doi: 10.1261/rna.725208

Figure Lengend Snippet: Genome-wide search for hnRNP L-regulated alternative splicing targets: combined microarray/RNAi strategy. (A) hnRNP L and LL, two closely related RNA-binding proteins. The domain structures of hnRNP L (P14866; 558 amino acids) and LL (Q53T80; 542 amino acids) are schematically represented (three canonical RRM motifs as orange boxes; glycine- and proline-rich regions in blue and green, respectively). (B) Outline of microarray/RNAi strategy. (C,D) RNAi knockdown of hnRNP L and LL: validation by (C) quantitative RT-PCR and (D) Western blotting. HeLa cells were treated with siRNA oligonucleotides specific for hnRNP L, LL, both L and LL, or as a control, luciferase mRNAs. (C) Relative mRNA levels are diagrammed (filled bars, hnRNP L; striped bars, hnRNP LL), normalized to luciferase. (D) Lysates were prepared after knockdowns (as indicated above the lanes), and hnRNP L (left panel), hnRNP LL (right panel), and as internal standard, γ-tubulin (lower panels) were detected by Western blotting. (E) Growth curves of HeLa cell cultures after RNAi knockdown of hnRNP L, LL, and L/LL double knockdown. HeLa cell cultures were treated with siRNA oligonucleotides specific for hnRNP L (ΔL; red squares), hnRNP LL (ΔLL; green triangles), or both L and LL (ΔL + ΔLL; blue crosses); luciferase knockdown served as a control (Δluc; black diamonds). After 24, 48, 72, and 96 h, cell densities were measured. (F) Alternative splicing of endogenous GSTZ1 mRNA. Total RNA was prepared after knockdown in HeLa cells (as indicated above the lanes), and alternative splicing of endogenous GSTZ1 mRNA (exon 5 skipping, as schematically shown on the right) was assayed by semiquantitative RT-PCR; exon skipping is quantitated as a percentage below. (M) DNA size markers (DNA ladder mix; Fermentas).

Article Snippet: Total RNA was prepared after knockdown in HeLa cells (as indicated above the lanes), and alternative splicing of endogenous GSTZ1 mRNA (exon 5 skipping, as schematically shown on the right ) was assayed by semiquantitative RT-PCR; exon skipping is quantitated as a percentage below. (M) DNA size markers (DNA ladder mix; Fermentas). illustrates our general strategy of searching for alternative splicing targets of hnRNP L and LL: Either hnRNP L, LL, or both in combination were down-regulated in HeLa cell culture by specific double-stranded siRNAs, with luciferase knockdown serving as a control; each of these RNAi knockdown reactions was carried out in three biological replicates.

Techniques: Genome Wide, Alternative Splicing, Microarray, RNA Binding Assay, Knockdown, Biomarker Discovery, Quantitative RT-PCR, Western Blot, Control, Luciferase, Reverse Transcription Polymerase Chain Reaction

Journal:

Article Title: Diverse roles of hnRNP L in mammalian mRNA processing: A combined microarray and RNAi analysis

doi: 10.1261/rna.725208

Figure Lengend Snippet: Combined microarray/RNAi approach: Detection and validation of intron retention cases. Detection of intron retention targets (upper panels). The diagrams show log2 ratios of probe-set signal intensities from the microarray data across the DAF gene, each relative to the luciferase control values. For each probe set (X-axis), three values are given (Y-axis: ΔL, knockdown of hnRNP L, in red; ΔLL, knockdown of hnRNP LL, in green; ΔL + ΔLL, knockdown of both hnRNP L and LL, in black). Probe-set positions in the retained intron and flanking regions are shown below. RT-PCR validation of intron retention in DAF (NM_000574.2) and STRA6 (NM_022369.2) genes (lower panels). Total RNA was prepared after knockdown in HeLa cells (as indicated above the lanes), and alternative splicing of endogenous DAF and STRA6 mRNAs was measured by RT-PCR. The percentages of intron retention are listed with standard deviations (n = 3) below the corresponding lanes. (M) DNA size markers (DNA ladder mix; Fermentas).

Article Snippet: Total RNA was prepared after knockdown in HeLa cells (as indicated above the lanes), and alternative splicing of endogenous GSTZ1 mRNA (exon 5 skipping, as schematically shown on the right ) was assayed by semiquantitative RT-PCR; exon skipping is quantitated as a percentage below. (M) DNA size markers (DNA ladder mix; Fermentas). illustrates our general strategy of searching for alternative splicing targets of hnRNP L and LL: Either hnRNP L, LL, or both in combination were down-regulated in HeLa cell culture by specific double-stranded siRNAs, with luciferase knockdown serving as a control; each of these RNAi knockdown reactions was carried out in three biological replicates.

Techniques: Microarray, Biomarker Discovery, Luciferase, Control, Knockdown, Reverse Transcription Polymerase Chain Reaction, Alternative Splicing

Journal:

Article Title: Diverse roles of hnRNP L in mammalian mRNA processing: A combined microarray and RNAi analysis

doi: 10.1261/rna.725208

Figure Lengend Snippet: Alternative poly(A) site selection. Target detection of alternative polyadenylation in ASAH1 (NM_004315.2) (left panel; legends for X- and Y-axes as in Fig. 3). Probe-set positions in exon 5, intron 6, and exon 6 region of ASAH1 mRNA are shown below, together with positions and directions of the primers used [three gene-specific primers by arrows; oligo(dT) primer]. RT-PCR validation (right panels). Total RNA was prepared after knockdown in HeLa cells (as indicated above the lanes), and splicing/alternative polyadenylation of endogenous ASAH1 mRNA was assayed by RT-PCR. (Upper gel photo) oligo(dT) primer combined with exon 5 forward primer, reflecting use of internal poly(A) site. (Lower gel photo) Combination of three gene-specific primers as shown on the left, resulting in two products that reflect spliced mRNA and internally polyadenylated mRNA (as indicated on the side). The percentages of internal polyadenylation are given below the corresponding lanes (signal of lower band compared to the sum of both bands; with standard deviations, n = 3; since different reverse primers are used, these values do not yield absolute numbers on the mRNA variants but allow the comparison between the control and knockdown samples). (M) DNA size markers (DNA ladder mix; Fermentas).

Article Snippet: Total RNA was prepared after knockdown in HeLa cells (as indicated above the lanes), and alternative splicing of endogenous GSTZ1 mRNA (exon 5 skipping, as schematically shown on the right ) was assayed by semiquantitative RT-PCR; exon skipping is quantitated as a percentage below. (M) DNA size markers (DNA ladder mix; Fermentas). illustrates our general strategy of searching for alternative splicing targets of hnRNP L and LL: Either hnRNP L, LL, or both in combination were down-regulated in HeLa cell culture by specific double-stranded siRNAs, with luciferase knockdown serving as a control; each of these RNAi knockdown reactions was carried out in three biological replicates.

Techniques: Selection, Reverse Transcription Polymerase Chain Reaction, Biomarker Discovery, Knockdown, Comparison, Control

Journal:

Article Title: Diverse roles of hnRNP L in mammalian mRNA processing: A combined microarray and RNAi analysis

doi: 10.1261/rna.725208

Figure Lengend Snippet: Exon skipping and inclusion. Target detection in TJP1 (NM_003257.2), FALZ (NM_004459.5), PARK7 (NM_007262.3), MYL6 (NM_021019.2), FAM48A (NM_017569.2), and PAPOLA (NM_032632.3) mRNAs (upper panels; legends for X- and Y-axes as in Fig. 3). For each gene, probe-set positions in the cassette exon and its flanking regions are shown below. In case of FAM48A, the other probe set with a significant low dR value was filtered out, because its P-value was too high. (Lower panels) RT-PCR validation. Total RNA was prepared after knockdown in HeLa cells (as indicated above the lanes), and alternative splicing of the six endogenous genes assayed by RT-PCR. Note that for TJP1 the additional minor band (asterisk) above the lower band represents an RT-PCR product due to mispriming within exon 20 (data not shown). The percentages of exon inclusion are listed with standard deviations (n = 3) below the corresponding lanes. (M) DNA size markers (DNA ladder mix; Fermentas).

Article Snippet: Total RNA was prepared after knockdown in HeLa cells (as indicated above the lanes), and alternative splicing of endogenous GSTZ1 mRNA (exon 5 skipping, as schematically shown on the right ) was assayed by semiquantitative RT-PCR; exon skipping is quantitated as a percentage below. (M) DNA size markers (DNA ladder mix; Fermentas). illustrates our general strategy of searching for alternative splicing targets of hnRNP L and LL: Either hnRNP L, LL, or both in combination were down-regulated in HeLa cell culture by specific double-stranded siRNAs, with luciferase knockdown serving as a control; each of these RNAi knockdown reactions was carried out in three biological replicates.

Techniques: Reverse Transcription Polymerase Chain Reaction, Biomarker Discovery, Knockdown, Alternative Splicing

Journal:

Article Title: Diverse roles of hnRNP L in mammalian mRNA processing: A combined microarray and RNAi analysis

doi: 10.1261/rna.725208

Figure Lengend Snippet: Suppression of multiple exons. Target detection in ARGBP2 (NM_021069.2) and LIFR mRNAs (NM_002310.2) (upper panels; legends for X- and Y-axes as in Fig. 3). Probe-set positions in the long intron and its flanking regions, as well alternative splicing patterns of ARGBP2 and LIFR mRNAs are shown below. (Lower panels) RT-PCR validation. Total RNA was prepared after knockdown in HeLa cells (as indicated above the lanes), and alternative splicing of endogenous ARGBP2 and LIFR mRNAs was assayed by RT-PCR. The identities of the RT-PCR products (as determined by sequence analysis) are diagrammed on the right. In each case, the percentages of exon inclusion (all internal exons combined) are listed below the corresponding lanes. (M) DNA size markers (DNA ladder mix; Fermentas).

Article Snippet: Total RNA was prepared after knockdown in HeLa cells (as indicated above the lanes), and alternative splicing of endogenous GSTZ1 mRNA (exon 5 skipping, as schematically shown on the right ) was assayed by semiquantitative RT-PCR; exon skipping is quantitated as a percentage below. (M) DNA size markers (DNA ladder mix; Fermentas). illustrates our general strategy of searching for alternative splicing targets of hnRNP L and LL: Either hnRNP L, LL, or both in combination were down-regulated in HeLa cell culture by specific double-stranded siRNAs, with luciferase knockdown serving as a control; each of these RNAi knockdown reactions was carried out in three biological replicates.

Techniques: Alternative Splicing, Reverse Transcription Polymerase Chain Reaction, Biomarker Discovery, Knockdown, Sequencing

Journal: Methods in molecular biology (Clifton, N.J.)

Article Title: Discovery of RNA Binding Small Molecules Using Small Molecule Microarrays

doi: 10.1007/978-1-4939-6584-7_11

Figure Lengend Snippet: Overview of SMM strategy to identify RNA-binding small molecules. Small molecule libraries prepared in 384-well plates are utilized to print onto isocyanate-functionalized slides by the microarrayer. Prepared SMMs are next incubated with the labeled RNA of interest and scanned to detect hits

Article Snippet: Printing of Small Molecule Microarrays MicroGrid II Microarrayer (BioRobotics) or equivalent.

Techniques: RNA Binding Assay, Incubation, Labeling