Gene Functional Study Of Key Factors(RIOK-1,RIOK-2 And DIM1) In Maturation Of 40S Pre-rRNA From Strongyloides Stercoralis

As a ribonucleoprotein(RNP)complexes,ribosomes are the cellular machines that translate m RNAs into proteins in all cells in Nature.In eukaryotes,ribosome assembly is a complex process that requires the concerted actions of > 200 assembly factors and several energy-consuming enzymes transiently associated with the assembling ribosome to promote the processing and folding of r RNA and the binding of ribosomal proteins,thereby generates mature,translation-competent ribosomal subunits.The cleavage at the3? end of the 18 S r RNA is one of the final events in small ribosomal subunit biogenesis that generates mature small ribosomal subunits.The deletion of non-ribosomal protein RIOK-1 or RIOK-2 protein kinases involved in this process causes disturbances in the ribosome maturation process.RIOK-1 and RIOK-2 are protein kinases necessary for 40 S pre-r RNA maturation.In addition,they are shown to drive proliferation and survival,and their expressions are linked to oncogenic AKT signaling.Another processing factor that plays a key role in the maturation of small ribosomal subunit-DIM1 is a substrate for the RIOK-2 protein kinase,which is required for the pre-processing reaction to synthesize the small ribosomal 18 S r RNA by methylating the ribosomal RNA.Although the functions of RIOK-1,RIOK-2 and DIM1 have been elucidated in yeast and mammalian cells,little is known about their roles in parasites.In the present study,extending from the previous work,the functions of these molecules in parasitic nematode,Stongyloides stercoralis,have been explored using transgenesis combined with the mutation technique.The following findings were obtained in the present study:1)Ss-RIOK-1 is essential for the development and survival of S.stercoralis larvaeSs-RIOK-1 expressed in the cytoplasm of neurons and some hypodermal cells in the wild-type strain(UPD)of S.stercoralis contains the conserved catalytic domain.Larvae expressing the dominant negative mutant Ss-RIOK-1 that lost the catalytic activity had a severe defect in development,and eventually died during the development from the free-living first-stage larvae(PFL L1)to the second-stage larvae(PFL L2).As predictable,the wild-type Ss-RIOK-1 can effectively rescue this defect.The findings demonstrated that Ss-RIOK-1 is essential for the development and survival of S.stercoralis free-living larvae,and that catalytic activity is essential for its function.2)Ss-RIOK-2 regulates larval growth and development and the ATP-binding ability of RIOK-2 is essential for S.stercoralis egg hatchingSs-RIOK-2 was mainly expressed in the cytoplasm of intestinal and hypodermalcells in transgenic larvae and along with the development of larvae from the PFL L1 to the infective third-stage larvae(i L3),the number of the larvae expressing Ss-RIOK-2 in the hypodermis increased,while those in the intestine decreased.The mutation of 228 th Asp at the catalytic domain of Ss-RIOK-2 delayed the growth and development in transgenic larvae.Interestingly,the mutation at 123 th in the ATP-binding domain of Ss-RIOK-2 caused an egg hatching failure along with the change of its localization from the cytoplasm to the nucleus in transgenic eggs,and the wild-type Ss-RIOK-2 can effectively rescue this defect.Our findings demonstrated that the ATP-binding ability but not the catalytic activity of Ss-RIOK-2 is essential for the egg hatching of S.stercoralis.3)Molecular characterization of Ss-dim-1,and the expression pattern of Ss-DIM1 in S.stercoralis larvaeThe c DNA sequence of Ss-dim-1 gene of S.stercoralis was obtained from the NCBI and Wormbase database.The coding region was 915 bp in length,encoding a predicted protein of 304 amino acids containing a highly conserved functional domain.Moreover,Ss-DIM1 was significantly phosphorylated by Ss-RIOK-2 protein kinase in vitro.RNAseq revealed that Ss-dim-1 is transcribed throughout all developmental stages with higher transcript abundances in parasitic and free-living adult stages.The transcriptional reporter expression driven by the predicted promoter sequence showed that Ss-DIM1 was expressed in gonad primordium.In this thesis,the roles of Ss-RIOK-1 and Ss-RIOK-2 were demonstrated in worm growth and development and egg hatching.In addition,DIM1 methyltransferase was isolated and identified from S.stercoralis,and its expression profile in larvae was detected.The findings from the thesis provide a basis for exploring the functional roles of these molecules in regulating the small ribosomal subunit maturation.