Evolutionarily conserved exon definition interactions with U11 snRNP mediate alternative splicing regulation on U11–48K and U11/U12–65K genes

<div><p>Many splicing regulators bind to their own pre-mRNAs to induce alternative splicing that leads to formation of unstable mRNA isoforms. This provides an autoregulatory feedback mechanism that regulates the cellular homeostasis of these factors. We have described such an autoregulatory mechanism for two core protein components, U11–48K and U11/U12–65K, of the U12-dependent spliceosome. This regulatory system uses an atypical splicing enhancer element termed USSE (U11 snRNP-binding splicing enhancer), which contains two U12-type consensus 5′ splice sites (5′ss). Evolutionary analysis of the USSE element from a large number of animal and plant species indicate that USSE sequence must be located 25–50 nt downstream from the target 3′ splice site (3′ss). Together with functional evidence showing a loss of USSE activity when this distance is reduced and a requirement for RS-domain of U11–35K protein for 3′ss activation, our data suggests that U11 snRNP bound to USSE uses exon definition interactions for regulating alternative splicing. However, unlike standard exon definition where the 5′ss bound by U1 or U11 will be subsequently activated for splicing, the USSE element functions similarly as an exonic splicing enhancer and is involved only in upstream splice site activation but does not function as a splicing donor. Additionally, our evolutionary and functional data suggests that the function of the 5′ss duplication within the USSE elements is to allow binding of two U11/U12 di-snRNPs that stabilize each others' binding through putative mutual interactions.</p></div

Antibody titration assays using AF-labeled or Eu-labeled protein L.

<p><b>A</b>) Anti-GST antibody titrated against Eu-labeled GST-VP1u and AF-labeled protein L. <b>B</b>) Anti-SA antibody titrated against Eu-labeled SA and AF-labeled protein L. <b>C</b>) Anti-SA antibody titrated against AF-labeled SA and Eu-labeled protein L. <b>D</b>) Anti-GST antibody titrated against AF-labeled GST-VP1u and Eu-labeled protein L. In all setups antibody concentrations were from 3.1 nM to 50 nM, and the antigen and protein concentration was constant (20 nM). Anti-GST antibody was used as a control for SA assays, and anti-SA for GST assays. The third line represents a background control with no antibody. The y-axis represents response counts obtained from Victor² fluorometer. The error bars represent ± standard deviation between parallel wells.</p

Antibody titration in ELISA.

<p>Monoclonal <b>A</b>) SA and <b>B</b>) GST antibodies were titrated in SA- or GST coated ELISA. The error bars represent ± standard deviation between parallel wells.</p

The performance of Fab-fragments generated from anti-SA MAb in the protein L assay.

<p>Fab-fragments were diluted from 200 nM to 50 nM, and a dilution series (50 nM to 12.5 nM) of anti-SA MAb acting as control was tested in parallel. The third line represents a control for fluorescence background induced by AF-labeled protein L and Eu-SA. The y-axis represents response counts obtained from Victor² fluorometer. The error bars represent ± standard deviation between parallel wells.</p

Eu- and AF-labeled protein L titration assays.

<p>AF-labeled protein L was titrated (100–6.3 nM) separately with <b>A</b>) Eu-labeled GST-VP1u and <b>B</b>) Eu-labeled SA. Eu-labeled protein L was titrated (100–6.3 nM) separately with <b>C</b>) AF-labeled SA and <b>D</b>) AF-labeled GST-VP1u. The anti-GST antibody was used as control for SA and anti-SA antibody for GST-VP1u. The third line represents a background control without antibody. The y-axis represents response counts obtained from Victor fluorometer. The error bars represent ± standard deviation between parallel wells.</p

TR-FRET signals induced by anti-SA antibody diluted in human IgG at various concentrations.

<p>Anti-SA Antibody was diluted in human IgG 1/4, 1/8 and 1/16, and tested with two dilutions of AF-labeled protein L: 100 nM and 200 nM, while the antigen concentration was kept constant (10 nM). Dilutions of anti-human IgG (80 nM), anti-SA (20 nM) were used as antibody controls, and buffer with AF-L and Eu-SA at 10 nM was used as background control. The y-axis represents response counts obtained from Victor² fluorometer. The error bars represent ± standard deviation between parallel wells.</p

Size determination of immune complexes in density gradient ultracentrifugation.

<p>Immune complexes were formed by mixing A) Monoclonal anti-SA, Eu-SA and AF647-SA at 8∶1:1 ratio B) Monoclonal anti-SA, Eu-SA and AF647-SA at 2∶1:1 ratio C) Polyclonal anti-SA (Abcam), Eu-SA and AF647-SA at 3∶1:1 ratio D) Polyclonal anti-SA (Springbioscience) at 3∶1:1 ratio, and separated by ultracentrifugation in preformed 0–70% sucrose gradient. Fractions collected from bottom were analyzed for TR-FRET activity, sucrose concentration and antibody concentration. The black line represents relative TR-FRET value (each value divided by the highest measured value) and the dark grey line represents relative antibody concentration (normalized to the highest value as above) in each fraction. Estimated molecular weight in kilodaltons (Y-axis) is calculated according to each sucrose concentration (X-axis) and shown in light grey line. The second y-axis is a relative scale (from 0–100% of the TR-FRET in fraction with highest TR-FRET signal) used for both TR-FRET signal and antibody amount.</p

End point titration of the antibodies and testing of Fab fragments.

<p>A) Polyclonal antibody versus monoclonal antibody end point titration (17 nM to 0.016 nM) using ELISA. B) A dilution series (0.09 nM to 120 nM) of Fab fragments was tested in SA-ELISA, using monoclonal antibody (0.03 nM to 40 nM) as control. Error bars are ± standard deviation calculated from parallel wells.</p

A schematic overview of the experimental procedures.

<p>A) Solution-phase assay setup. All reagents were pipetted onto a 384-well plate and the TR-FRET signal read directly. B) One-step solid-phase assay. The antigen mixture and the antibody pipetted onto protein A coated wells and the TR-FRET signal is measured directly (measurement 1) and after replacing the mixture with TBS (measurement 2). C) Two-step solid-phase assay. In the first step the antibody was allowed to bind to a protein A coated well. In the second step equimolar antigen mixture was added. The TR-FRET signal was measured directly after antigen addition (measurement 1) and after replacing the antigen mixture with TBS (measurement 2).</p

Antibody vs. antigen titration.

<p>A) Polyclonal anti-SA antibody (4.16 nM to 66.6 nM), B) polyclonal anti-GST antibody (2.08 nM to 33.3 nM) and C) monoclonal anti-SA antibody (4.16 nM to 66.6 nM) were mixed with five different concentrations of Eu-SA and AF647-SA (at equimolar ratio), and resulting TR-FRET was measured. Dilution series of Fab fragments (7.5 nM to 120 nM) D) tested with 8 nM antigens (Eu-SA and AF647-SA). The TR-FRET (expressed in response counts) is the average of two measurements. Raw data are presented on the left panel, and the normalized data on the right panel. The concentration of specific IgG (for PAb), is indicated in brackets. Error bars are ± standard deviation calculated from parallel wells.</p