Complex splicing control of the human Thrombopoietin gene by intronic G runs

Abstract

The human thrombopoietin (THPO) gene displays a series of alternative splicing events that provide valuable models for studying splicing mechanisms. The THPO region spanning exon 1-4 presents both alternative splicing of exon 2 and partial intron 2 (IVS2) retention following the activation of a cryptic 3' splice site 85 nt upstream of the authentic acceptor site. IVS2 is particularly rich in stretches of 3-5 guanosines (namely, G1-G10) and we have characterized the role of these elements in the processing of this intron. In vivo studies show that runs G7-G10 work in a combinatorial way to control the selection of the proper 3' splice site. In particular, the G7 element behaves as the splicing hub of intron 2 and its interaction with hnRNP H1 is critical for the splicing process. Removal of hnRNP H1 by RNA interference promoted the usage of the cryptic 3' splice site so providing functional evidence that this factor is involved in the selection of the authentic 3' splice site of THPO IVS2.

Figure 1

Comparison of splicing pattern of endogenous THPO and IVS2-wt construct. (a) Analysis of the exon 1-exon 4 splicing pattern by RT–PCR of endogenous THPO gene constitutively expressed in Hep3B and HEK293 cell lines. THPOwt is the full length transcript (exon 1+2+3+4). THPO+85 is the full length transcript including 85 nt from IVS2. THPO-Ex2 is the mRNA excluding exon 2. H₂O indicates the PCR control. M indicates the lane with 1 Kb plus DNA marker. (b) Nucleotide sequence encompassing intron 1-exon 2- intron 2-exon 3 of human THPO gene. Introns are shown in lower case, exons are in bold upper case. The score of acceptor (a=) and donor (d=) splice sites calculated using the Splice Site Prediction by Neural Network program (SSPNN, ) are indicated. The ten G runs (in uppercase) within intron 2 are numbered 1–10 and the mutagenesis carried out for each G runs is shown in brackets. An arrow indicates the cryptic 3′ splice site within IVS2. (c) Scheme of the human THPO minigene used for transfection experiments. White boxes correspond to the human THPO exons (Ex) 1–4 and thin lines represent introns (IVS). Sizes of exons and introns are shown. (d) Analysis of pre-mRNA splicing of IVS2-wt and IVS2-G1-G10m constructs. Amplicons were separated on a 1.3% (w/v) agarose gel.

Figure 2

Mapping of the G runs splicing control elements within THPO IVS2. (a) and (c) Schemes of the constructs used for transient transfections. The presence of G runs is indicated by its position-number. (b) and (d) Splicing pattern analyses of the RT–PCR products derived from cellular RNA, separated on a 1.3% agarose gel and stained by ethidium bromide. THPOwt is the full length transcript (exon 1+2+3+4). THPO+85 is the full length transcript including 85 nt from IVS2. THPO-Ex2 is the mRNA excluding exon 2. THPO+85-Ex2 indicates the mRNA species carrying the 85 nt inclusion along with exon 2 skipping.

Figure 3

Effects of combined G7, G8 and G9 runs on THPO splicing. (a) and (b) Schemes of the constructs used for transient transfections. The presence of G runs is indicated by its position-number. (c) and (d) Splicing pattern analyses of the RT–PCR products derived from cellular RNA, separated on a 1.3% agarose gel and stained by ethidium bromide.

Figure 4

Role of G7 run and its flanked nucleotides in the 3′ splice site selection within IVS2. (a) Scheme of the constructs used for transient transfections. (b) RT–PCR of the constructs carrying G7-G8-G9 carrying the reduction from 4 to 3 G in the G7 element and with the flanked nucleotides mutated are indicated with G7g3 and G7pyr, respectively. (c) RT–PCR products derived from the constructs where the G7 was disrupted at the level of the G-core (IVS-wt-G7m) or in its flanking nucleotides (IVSwt-G7pyr). (d) The construct IVS2-G6-G7pyr-G8G9 shows a significant rescue of the THPOwt isoform (lane 1).

Figure 5

Interaction of nuclear proteins with G runs within THPO IVS2. (a) Sequence of oligos KpnI/Hind III cloned in pBS KS under T7 promoter control for in vitro transcription. G7, G8 and G9 runs are in uppercase and the position of G mutagenized by point mutations are indicated with a vertical line. (b) UV-cross-linking assay using HeLa nuclear extract with in vitro transcribed THPO789 and THPO789m RNAs. The arrow shows the position of the 58 kDa complex. (c) Competition analysis confirms the binding specificity of the 58 kDa complex following addition of cold THPO789 and THPO789m RNAs to labeled THPO789 RNA in the presence of HeLa nuclear extract. The molar ratio of cold/labeled RNA was three and six. The arrows indicate the 58 kDa complex. (d) Coomassie stained SDS–PAGE of a pull-down assay using adipic dehydrazide beads derivatized with the RNAs following incubation with HeLa nuclear extract. In the lane from the wild type RNA-derivatized beads (THPO789), the arrows indicate the proteins that are present only in the wild type THPO789 RNA-derivatized beads. (e) UV-cross-linking assay using HeLa nuclear extract with RNA oligos spanning only wild type G7 run (G7wt), G7 with its flanking nucleotides mutagenized (G7pyr) and of G7 with G-core disrupted (G7m). The arrow shows the position of the hnRNP H1 proteins.

Figure 6

Knock-down of hnRNPH1 causes the activation of the cryptic acceptor site within IVS2. (a) Western blot carried out with polyclonal anti-hnRNP H1 antiserum on HeLa cell non transfected (NT HeLa cells) or transfected with siRNA against hnRNP H1 (siRNA-H1) or luciferase (siRNA-luc). β-tubulin and α-actin were probed with polyclonal antibodies as a control of total protein loading. Relative amounts of hnRNP H1 silencing are indicated below each lane. Standard deviations were always ≤20%. (b) The panel shows splicing pattern analysis of RT–PCR products after cotransfection of IVS2 G7G8G9 and IVS2-G1-6m along with either the siRNA-H1 (lanes 2 and 4) or a control siRNA-luc oligonucleotides (lanes 1 and 3). (c) RT–PCR experiments after RNAi against hnRNP H1 and cotransfection with the IVS2-wt-G7m minigene. Lane 1 shows the cotransfection of IVS-wt-G7m with a control siRNA. After siRNA anti-hnRNP H the THPO+85 isoform became predominant (lane 2). Lower panels show the related anti-hnRNP H1 western blot analyses. β-tubulin was used as a control of total protein loading. Relative amount of hnRNP H1 silencing is indicated below each lane. Standard deviation were ≤20%. (d) RT–PCR experiments in siRNA-H1-treated in HeLa cells. Lanes 1 and 2 show the results of PCR after cotransfecting the IVS2-wt minigene. After the reduction in the expression of hnRNP H there is a small increase of the THPO+85 isoform. The control transfection was loaded in lane 1. Lower panels show the related anti-hnRNP H1 western blot analyses with indication of relative amounts of hnRNP H1 silencing. Standard deviations were ≤20%.

Figure 7

Evolutionary comparison of the splicing control elements found in THPO intron 2 in human, macaca, chimpanzee, dog, mouse and rat. (a) The alignment of intron 2 from different species was generated using the GeneBee method (). All G runs in human intron 2 are underlined. The human cryptic 3′ splice site between G8 and G9 runs is boxed. (b) Graphical phylogenetic tree generated by the GeneBee server that depicts the distances among different species for THPO IVS2.