Auto- and cross-regulation of the hnRNP L proteins by alternative splicing

Abstract

We recently characterized human hnRNP L as a global regulator of alternative splicing, binding to CA-repeat and CA-rich elements. Here we report that hnRNP L autoregulates its own expression on the level of alternative splicing. Intron 6 of the human hnRNP L gene contains a short exon that, if used, introduces a premature termination codon, resulting in nonsense-mediated decay (NMD). This "poison exon" is preceded by a highly conserved CA-rich cluster extending over 800 nucleotides that binds hnRNP L and functions as an unusually extended, intronic enhancer, promoting inclusion of the poison exon. As a result, excess hnRNP L activates NMD of its own mRNA, thereby creating a negative autoregulatory feedback loop and contributing to homeostasis of hnRNP L levels. We present experimental evidence for this mechanism, based on NMD inactivation, hnRNP L binding assays, and hnRNP L-dependent alternative splicing of heterologous constructs. In addition, we demonstrate that hnRNP L cross-regulates inclusion of an analogous poison exon in the hnRNP L-like pre-mRNA, which explains the reciprocal expression of the two closely related hnRNP L proteins.

FIG. 1.

Exon-intron structure, nucleotide sequence conservation, and map of CA-rich elements in the hnRNP L gene. (A and B) Nucleotide sequence conservation is diagrammed for the hnRNP L gene from 28 vertebrate species (UCSC Genome browser; http://genome.ucsc.edu/) (A) and by comparing human versus mouse sequences, plotting percentage of identity over 50-nt windows across the gene (B). Blue regions indicate the 13 exons; the highly conserved region in intron 6 is circled in red. (C and D) Exon-intron structure of human (C) and mouse (D) hnRNP L genes. Above the line, red bars represent hnRNP L binding motifs, with their height corresponding to their score (for how these scores were derived, see reference 16).

FIG. 2.

Exon 6A in the human hnRNP L gene is an NMD target. (A) HnRNP L exon 6A inclusion is increased after cycloheximide treatment. HeLa cells were treated with cycloheximide over 3 h, and total RNA was prepared after 0, 0.5, 1, and 3 h (as indicated) and analyzed by semiquantitative RT-PCR for exon 6A inclusion (top). Quantitation of exon 6A inclusion is given below the lanes (in percentages). The panels below show control RT-PCRs for β-actin mRNA (middle), and for alternative splicing of another hnRNP L target gene, TJP1 (bottom; the band marked by the asterisk represents a PCR artifact). RT-PCR products are schematically represented on the right. (B and C) hnRNP L exon 6A inclusion is increased after UPF1 knockdown (ΔUPF1). UPF1 expression was downregulated in HeLa cells by RNAi, with a luciferase knockdown (Δluc) as a control (see panel B for Western blot analysis of UPF1 and control γ-tubulin protein levels). (C) Total RNA after knockdowns of UPF1 (ΔUPF1) and control luciferase (Δluc) was analyzed for exon 6A inclusion by RT-PCR (RT-PCR products schematically represented on the right). Quantitation of hnRNP L exon 6A inclusion is given below the lanes (in percentages). M, DNA size markers.

FIG. 3.

CA cluster in intron 6 binds hnRNP L, as well as hnRNP LL. Biotinylated 5′ and 3′ parts (lanes 2 and 3, respectively) of the CA cluster of hnRNP L intron 6, as well as biotinylated (CA)₃₂ RNA (lane 4) or an unrelated biotinylated control RNA (lane 5), were incubated in HeLa nuclear extract, followed by neutravidin-agarose selection, release of bound protein, and Western blot analysis with anti-hnRNP L monoclonal antibody 4D11 (top panel) or anti-hnRNP LL antibody (bottom panel). In a control reaction, no RNA was added (lane 6). For comparison, 10% of the nuclear extract input was analyzed (lane 1).

FIG. 4.

HnRNP L activates exon 6A inclusion. (A and B) HnRNP L expression was downregulated in HeLa cells by RNAi (ΔL), with luciferase (Δluc) as a control, and hnRNP L and control γ-tubulin protein levels were analyzed by Western blotting (panel A, compare lanes 1 and 2). After UPF1 and control luciferase knockdowns, with (+) or without (−) an additional cycloheximide treatment (cyclohex), total RNA was prepared and exon 6A inclusion was measured by RT-PCR (panel B, lanes 1 to 4; RT-PCR products are schematically represented on the right). M, DNA size markers. (C) RNAi-mediated downregulation of hnRNP L mRNA: real-time RT-PCR analysis. hnRNP L mRNA levels were quantitated after RNAi knockdown, using real-time RT-PCR and primer combinations in exons 2 and 3, 4 and 5, 7 and 8, 8 to 10, or 6A and 7, each normalized to β-actin mRNA levels and in comparison to the luciferase control knockdown (set at 100%; each primer pair was measured twice). Below, the percentages of hnRNP L mRNA downregulation are given, including the standard deviations. In addition, the exon 6A inclusion ratios were determined from these values, each relative to exons 2 and 3, 4 and 5, 7 and 8, or 8 to 10 (the percentages, including standard deviations are shown). (D) HnRNP L overexpression downregulates endogenous hnRNP L protein levels. HeLa cells were transfected with constructs expressing V5-His-tagged hnRNP L (L-V5-His) or GST-tagged hnRNP L (L-GST) or were mock transfected. At 2 days posttransfection endogenous hnRNP L (endo-L), as well as V5-His- and GST-tagged hnRNP L (see arrows), was detected by Western blotting, as well as γ-tubulin as an input control. The levels of endogenous hnRNP L were quantitated as a percentage relative to mock-treated samples and normalized to γ-tubulin levels.

FIG. 5.

Intronic CA cluster acts as an hnRNP L-dependent enhancer of hnRNP L exon 6A inclusion. (A) Exon-intron structure of human hnRNP L gene, with the red bars above the line indicating hnRNP L binding motifs (height of bars representing CA richness [16]). The 3′-CA cluster region and exon 6A (boxed area) was inserted in pDUP, to give the heterologous construct T7-DUP 3′-CA cluster-6A. As a control, the 3′-CA cluster was replaced by an unrelated sequence (T7-DUP control-6A). (B and C) HnRNP L- and CA cluster-dependent activation of exon 6A inclusion in vitro. HeLa nuclear extract (NE; panel B, lane 1) was depleted of hnRNP L (NEΔL; see lane 3) or mock depleted (NEΔmock; lane 2) and analyzed by Western blotting with antibody against hnRNP L (panel B, top) or as a control, GAPDH (panel B, bottom). (C) The 3′ CA-cluster-6A pre-mRNA was spliced for 120 min in L-depleted nuclear extract complemented with increasing amounts of recombinant hnRNP L (0 to 400 ng per 25 μl, as indicated; see lanes 1 to 7) or in mock-depleted nuclear extract (lane 8). The control pre-mRNA (control-6A) was spliced in L-depleted extract (NEΔL; lane 9), in L-depleted extract complemented with 200 ng of hnRNP L per 25-μl reaction mixture (NEΔL+L; lane 10), and in mock-depleted extract (NEΔmock; lane 11). Splicing was monitored by RT-PCR, the positions of pre-mRNAs and inclusion/skipping products schematized on the right (the asterisk marks a partially spliced product, with the first intron retained). M, DNA size markers. (D) CA cluster-dependent exon 6A inclusion in vivo. HeLa cells were mock transfected (mock; lane 1) or transfected with the 3′-CA cluster-6A construct (lane 2) or with the control-6A construct (lane 3). At 2 days posttransfection total RNA was prepared and analyzed by RT-PCR, monitoring exon 6A inclusion. The positions of inclusion and skipping products are schematized on the left. M, DNA size markers. (E) hnRNP l- and CA cluster-dependent activation of exon 6A inclusion in a two-exon context in vitro. The two-exon pre-mRNAs 3′CA cluster-exon6A/+77 (lanes 1 to 6) or control-exon6A/+77 (lanes 7 to 12), each with 77 nt of intron 6A, were spliced in nuclear extract (NE; for 0, 60, and 120 min, as indicated), in L-depleted (NEΔL; 120 min), or in mock-depleted nuclear extract (NEΔmock; 120 min). For complementation with recombinant hnRNP L (+L), 200 ng of per 25-μl reaction mixture were used. Splicing was monitored by RT-PCR, the positions of pre-mRNAs and spliced product are indicated on the right. M, DNA size markers. (F) Exon 6A recognition depends on intact 5′ splice site. The two-exon pre-mRNAs 3′CA cluster-exon6A/+14 (with 14 nucleotides of intron 6A), carrying a wild-type (lanes 1 to 4) or mutated 5′ splice site (lanes 5 to 8), were spliced in nuclear extract for the times indicated (in minutes). Splicing was monitored by RT-PCR, and the positions of pre-mRNAs and spliced product are indicated on the right. M, DNA size markers.

FIG. 6.

Cross-regulation of the paralog gene hnRNP LL by hnRNP L. (A and B) Exon 6A in the human hnRNP LL pre-mRNA is an NMD target. (A) hnRNP LL exon 6A inclusion is increased after cycloheximide treatment. HeLa cells were treated with cycloheximide over 4 h, and total RNA was prepared after 0, 0.5, 2, 3, and 4 h (as indicated) and analyzed by semiquantitative RT-PCR for exon 6A inclusion (top) and, as a control, for β-actin mRNA (bottom). RT-PCR products are schematically represented on the right for both panels. (B) hnRNP LL exon 6A inclusion is increased after UPF1 knockdown. UPF1 expression was downregulated in HeLa cells by RNAi (ΔUPF1), with a luciferase knockdown as a control (Δluc). Total RNA after UPF1 and control luciferase knockdowns was analyzed for hnRNP LL exon 6A inclusion by RT-PCR. Quantitation of hnRNP LL exon 6A inclusion is given below the lanes (in percentages). M, DNA size marker. (C) hnRNP L activates hnRNP LL exon 6A inclusion. hnRNP L expression was downregulated in HeLa cells by RNAi (ΔL), with a luciferase knockdown (Δluc) as a control, with (+) or without (−) an additional cycloheximide treatment after knockdown (cyclohex). Total RNA after UPF1 and control luciferase knockdowns was analyzed for hnRNP LL exon 6A inclusion by RT-PCR (products schematically represented on the right). Quantitation of hnRNP LL exon 6A inclusion is given below the lanes (in percentages). M, DNA size markers.

FIG. 7.

Model of hnRNP L auto- and cross-regulation. The hnRNP L pre-mRNA can switch in its exon 6-7 region between two alternative splicing modes (right part of the figure). First, at low hnRNP L levels, exon 6A is skipped, resulting in functional hnRNP L mRNA and thereby increasing L protein levels (top). Second, at high levels hnRNP L binds to the intronic CA-cluster enhancer, promoting exon 6A inclusion, which results in NMD-mediated downregulation of hnRNP L expression (bottom). This hnRNP L-dependent switch creates an autoregulatory feedback mechanism, which contributes to hnRNP L homeostasis. In addition, hnRNP L regulates diverse alternative splicing processes in many other target genes (13, 16), among them (see left part of figure) the paralog gene hnRNP LL, which contains an analogous poison exon 6A that also switches, depending on hnRNP l—between inclusion and skipping. Finally, hnRNP LL targets many alternative splicing processes in a cell-type-specific manner (27).