KHSRP ameliorates acute liver failure by regulating pre-mRNA splicing through its interaction with SF3B1

Acute liver failure (ALF) is characterized by the rapidly progressive deterioration of hepatic function, which, without effective medical intervention, results in high mortality and morbidity. Here, using proteomic and transcriptomic analyses in murine ALF models, we found that the expression of multiple splicing factors was downregulated in ALF. Notably, we found that KH-type splicing regulatory protein (KHSRP) has a protective effect in ALF. Knockdown of KHSRP resulted in dramatic splicing defects, such as intron retention, and led to the exacerbation of liver injury in ALF. Moreover, we demonstrated that KHSRP directly interacts with splicing factor 3b subunit 1 (SF3B1) and enhances the binding of SF3B1 to the intronic branch sites, thereby promoting pre-mRNA splicing. Using splicing inhibitors, we found that Khsrp protects against ALF by regulating pre-mRNA splicing in vivo. Overall, our findings demonstrate that KHSRP is an important splicing activator and promotes the expression of genes associated with ALF progression by interacting with SF3B1; thus, KHSRP could be a possible target for therapeutic intervention in ALF.


Hydrodynamic tail-vein injection
Hydrodynamic injection was performed as previously described (1,2).Briefly, constructed pcDNA3.1-Khsrp(contain complete mouse Khsrp CDS) and pcDNA3.1 were suspended in saline solution and subsequently injected into the lateral tail veins of male or female mice (0.1 ml/g body weight) in less than 7 seconds.The information of pcDNA3.1-Khsrpwas listed in Supplemental Table 2.

Western blot
Proteins were extracted from cells or tissues with RIPA lysis buffer (Servicebio) and quantified by the Bradford method (Sangon).Cell lysates were then loaded in SDS-PAGE gel to separate proteins and transferred to nitrocellulose membrane.

Serum measurements
The levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in serum were measured by using ALT Assay Kit and AST Assay Kit (Nanjing Jiancheng Bioengineering Institute).

Plasmids and short hairpin RNAs
All the plasmids used in this paper were generated using the Gibson assembly cloning method.The cDNA sequences of KHSRP CDS full length, KHSRP CDS

Generation of KHSRP knockdown and overexpression cells
For lentiviral stock preparation, 293T cells were cotransfected with pHAGE-KHSRP-FL or shKHSRP plasmid and three packaging plasmids (pGag-Pol, pRev, and pVSVG).Supernatants containing packaged lentivirus were collected after 48 h, passed through a 0.45 m filter and added to HL7702 cells along with 1 mg/ml polybrene (Sigma, H9268).Cells were selected using purinomycin (1g/ml).

Luciferase assay
Transient transfections of stable KHSRP overexpression cells or control cells were accomplished by plating cells at a density of 1× 10⁵ cells per well of a 24-well culture plate 24 h before transfection.The pMIR-GLO reporter vectors containing the 3′UTRs of TGF-β1, SF3B1, EGFR, PHF5A, and CDC25A were transfected using 1 μl of Lipofectamine 2000 transfection reagent (Invitrogen) following instructions.
After 24 h transfection, cells were harvested in Reporter lysis buffer (Progema), and the luciferase activity was determined by Luciferase assay system (Progema).The firefly luciferase activity was normalized to the Renilla luciferase activity.

Histology, Immunohistochemistry, and Apoptosis assay
Liver samples were fixed overnight in 4% formalin at room temperature, continuously dehydrated in ethanol, embedding with paraffin.Paraffin sections were stained with Meyer hematoxylin and stained with eosin.Representative areas of each stained tissue section were imaged at ×40 magnification.For immunohistochemistry, tissue sections were incubated with the antibodies against EGFR (510673, ZENBIO), SF3B3 (14577-1-AP, Proteintech), SF3B1 (sc-5a4655, Santa Cruz), Ki-67 (511390, ZENBIO) and PHF5A (15554-1-AP, Proteintech) for 1 h at room temperature.These slides were then subjected to horseradish peroxidase-linked secondary antibodies for 1 h at room temperature.The sections were visualized by using the DAB substrate kit (Vector Labs) and representative areas of each stained tissue section were imaged at ×100 magnification.ImageJ software was used to quantify the staining results.
Apoptosis was detected by TUNEL staining in liver tissues.TUNEL staining was performed using the TUNEL Assay kit (Elabscience) according to the manufacturer's instructions, with apoptotic cells exhibiting red nuclear fluorescence.Liver sections were counterstained with propidium iodide (DAPI) for 5-10 min to stain cell nuclei.
Finally, the fluorescent images were captured by using the Zeiss LSM 880 Confocal Microscope (Medical Research Center for Structural Biology, Wuhan University).

Co-immunoprecipitation (Co-IP)
A total of 1×10 6 cells for each immunoprecipitation were lysed with IP lysis buffer (Beyotime Biotechnology, Shanghai) on ice for 30 min and then sonicated in an ice-water bath.Cell lysates were incubated with the antibody overnight at 4°C.Before incubated with the cell lysate, the A/G agarose beads were treated with 5% BSA.The immunoprecipitates (IP) were analyzed for the presence of SF3B1 (sc-5a4655, Santa Cruz) and KHSRP (A302-021A, BETHYL) by Western blot.