Microprocessor mediates transcriptional termination of long noncoding RNA transcripts hosting microRNAs

Abstract

MicroRNAs (miRNAs) play a major part in the post-transcriptional regulation of gene expression. Mammalian miRNA biogenesis begins with cotranscriptional cleavage of RNA polymerase II (Pol II) transcripts by the Microprocessor complex. Although most miRNAs are located within introns of protein-coding transcripts, a substantial minority of miRNAs originate from long noncoding (lnc) RNAs, for which transcript processing is largely uncharacterized. We show, by detailed characterization of liver-specific lnc-pri-miR-122 and genome-wide analysis in human cell lines, that most lncRNA transcripts containing miRNAs (lnc-pri-miRNAs) do not use the canonical cleavage-and-polyadenylation pathway but instead use Microprocessor cleavage to terminate transcription. Microprocessor inactivation leads to extensive transcriptional readthrough of lnc-pri-miRNA and transcriptional interference with downstream genes. Consequently we define a new RNase III-mediated, polyadenylation-independent mechanism of Pol II transcription termination in mammalian cells.

Figure 1. Lnc-pri-miR-122 transcripts are capped but not polyadenylated

a. Gene map showing exon-intron structure and 3′ positioned pre-miR-122 hairpin. Position of primers and Northern probes are indicated. b. Northern blot showing spliced and unspliced lnc-pri-miR-122 from human liver and three cell lines as indicated. Levels of γ-actin mRNA and mature miR-122 were also measured. Note reduced levels of liver RNA were employed. c. Northern blot detecting unspliced and spliced lnc-pri-miR-122 in human liver RNA using intron and exon-specific probes. d. Levels of m⁷G-cap immunoselected RNA measured by RT-qPCR as indicated. e. Proportion of pA+ RNA relative to pA− for transcripts indicated measured by RT-qPCR, and for lnc-pri-miR-122 and γ-actin measured by northern blotting. Error bars represent s.d. of an average (n=3 independent experiments).

Figure 2. 3′ end mapping, subcellular distribution and rapid turnover of lnc-pri-miR-122

a. Gene map of lnc-pri-miR-122 showing position of primers for RT-qPCR and 3′ end mapping. b. PolyA polymerase (PAP)-dependent 3′ end mapping using 3′ RACE. Position of 3′ RACE PCR product shown by gel fractionation and location of mapped 3′ end cleavage products are shown by red arrow heads on the pre-miR-122 hairpin structure (see Supplementary Fig. 2). c. Pri-miR-122 and GAPDH mRNA distribution were determined between cell fractions (WC denotes whole cell, N nuclear and C cytoplasmic) by RT-PCR using indicated primers. Western blot shows purity of nuclear and cytoplasmic fractions by use of cytoplasmic and nuclear specific protein antibodies. d. RNA stability following actinomycin D inhibition of transcription at various time points measured by RT-qPCR of lnc-pri-miR-122 transcripts versus GAPDH mRNA. RNA levels are expressed relative to the levels at time=0, which were set to 1. Huh7 cells were used in all experiments. Error bars represent s.d. of an average (n=3 independent experiments).

Figure 3. Microprocessor defines transcriptional termination of lnc-pri-miR-122

a, b. Mapping nascent transcription across lnc-pri-miR-122 versus GAPDH in Huh7 cells. Gene maps show positions of amplicons used in analysis of chromatin RNA levels (left) and bromo UTP-labeled nuclear run on RNA (right). c. Western blot showing effective depletion of DGCR8 and CPSF-73 by siRNA transfection in Huh7 cells. Error bars represent s.d. of an average (n=3 independent experiments).

Figure 4. Microprocessor depletion leads to generation of pA− transcriptional readthrough products on lnc-pri-miR-122

a. RT-qPCR analysis of lnc-pri-miR-122 versus U6 snRNA or GAPDH mRNA controls using pA+ or pA− fractionated RNA from siRNA-treated Huh7 cells. pA+ RNA levels are expressed relative to pA− which were set to 1. Error bars represent s.d. of an average (n=3 independent experiments). b. Chromatin RNA-seq analysis of lnc-pri-miR-122 in control or DGCR8 siRNA-treated Huh7 cells. Red boxed 3′ region is shown below at higher magnification. Position of miRNA (miR-122) is shown by red vertical line.

Figure 5. Ectopically expressed lnc-pri-miR-122 switches to CPA when Microprocessor activity is inhibited

a. Schematic of lnc-pri-miR-122 expression construct driven under HIV-LTR promoter, with locations of northern probe, PCR amplicon and PAS (pA1). WT and Δ plasmids were generated with and without the pre-miR-122 hairpin. b. Northern blot showing that mature miR-122 is expressed from the WT, but not Δ, plasmid following transfection into HeLa cells. U6 snRNA is loading control. c. Northern blot showing spliced and unspliced lnc-pri-miR-122 transcripts generated from the WT plasmid transfected into HeLa cells are the same size as endogenous transcripts in Huh7 cells. d. Northern blot showing spliced and unspliced lnc-pri-miR-122 transcripts generated from the Δ plasmid are larger than the WT transcripts upon transfection into HeLa cells. e. Northern blot showing transcripts generated from the WT plasmid increase in size following Drosha or DGCR8 depletion in HeLa cells. f. RT-qPCR analysis of pA+ or pA− fractionated RNA extracted from siRNA-treated HeLa cells transfected with the WT plasmid. pA+ RNA levels of U6 snRNA, GAPDH mRNA and lnc-pri-miR-122 are expressed relative to pA− which were set to 1. Error bars represent s.d. of an average (n=3 independent experiments). g. Northern blot showing that mutation of PAS (pA1) does not affect migration of WT lnc-pri-miR-122, but leads to loss of unspliced and spliced transcripts generated from the Δ plasmid upon transfection into HeLa cells.

Figure 6. Microprocessor dependent chromatin RNA-seq profiles across pri-miRNA from HeLa cells

a. Reads (RPKM) across (MIR181A1HG) showing readthrough profiles following Microprocessor depletion. b. Metagene analysis of all expressed lnc-pri-miRNA. c. Reads across a protein coding gene (MCM7) harboring an intronic miRNA cluster showing intron accumulation following Microprocessor depletion d. Metagene analysis of all expressed protein coding genes harboring miRNAs. Direction of transcription indicated by green arrow and positions of miRNA by red vertical lines in a and c. Metagene profiles show transcription unit (between transcription start site, TSS and transcription end site, TES) followed by 10 kb of 3′ flanking region in b and d (Mann–Whitney U-test, P < 0.0001, two-tailed, n = 1400, for both cases). e. Western blot showing effective depletion of Drosha and DGCR8 by siRNA transfection in HeLa cells.

Figure 7. Microprocessor dependent termination prevents transcriptional interference

a. Chromatin RNA-seq profiles across MIR17HG-GPC5 tandem gene locus showing readthrough transcription following Microprocessor depletion. Black arrow and red box highlights reduction in GPC5 exon 1 peak following Microprocessor depletion. b. RT-qPCR analysis of chimeric transcripts versus GPC5 exon 1. Gene specific RT primer (arrowhead) in exon 1 and PCR amplicons are indicated by black bars. c. Chromatin RNA-seq profiles across convergent OGFRL1-LINC00472 gene locus. d. Read quantification for protein coding genes subject to transcriptional interference following Microprocessor depletion. e. mRNA levels of GPC5 and OGFRL1 were determined by RT-qPCR using exon specific primers. RNA levels are expressed relative to control siRNA which were set to 1. All values are normalized to GAPDH mRNA. f. Protein levels of GPC5 and OGFRL1 are reduced by transcriptional interference. Direction of transcription is indicated by green arrow and miRNA by red vertical lines. All experiments used HeLa cells. Error bars represent s.d. of an average (n=3 independent experiments).

Figure 8. Lnc-pri-miRNA may be CPA-incompetent or competent following Microprocessor depletion

RNA-seq of pA fractionated nuclear RNA from siRNA-treated HeLa cells. a. Nuclear RNA-seq profile of MIR17HG pA+ and pA− RNA following DGCR8 depletion. b. Nuclear RNA-seq profile of MIRLET7BHG pA+ and pA− RNA following DGCR8 depletion. Position of the PAS (pA) is marked by a dashed vertical line. Direction of transcription indicated by green arrow and positions of miRNA by red vertical lines in a and b. c. Model showing CPA in protein coding pri-miRNA allows generation of spliced mRNA and miRNA, while Microprocessor-driven termination in lnc-pri-miRNA generates miRNA and a pA− host transcript that is rapidly degraded. Lnc-pri-miRNA may be CPA-incompetent, leading to transcriptional readthrough and interference when Microprocessor cleavage is inhibited, or CPA-competent allowing effective transcription termination even in the absence of Microprocessor. Red thunderbolt with black fill depicts CPA-mediated cleavage. Blue thunderbolt with red fill depicts Microprocessor-mediated cleavage.