.1. People Of Acat1 Trans-splicing In The Maturation Of Mrna Processing And Its Molecular Mechanism. People Intracellular Protein N-terminal Myristoylation Modification System Vitro

Acyl-coenzyme A: cholesterol acyltransferase (ACAT) is an intracellularenzyme involved in cellular cholesterol homeostasis and in atherosclerotic foamcell formation. So far, two types of ACAT gene have been identified inmammals. In collaboration with professor TY Chang's Laboratory (inDepartment of Biochemistry, Dartmouth Medical School), we have found that thesequence corresponding to human ACAT1 cDNA K1 is produced from twodifferent chromosomes (Li BL et al. J. Biol. Chem. 274, 11060-71). It isspeculated that mRNA corresponding to human ACAT1 cDNA K1 is produced bya novel RNA recombination mechanism involving trans-splicing of two separateprecursor RNAs. This work mainly focuses on establishing the in vivo and invitro research systems of human ACAT1 mRNA trans-splicing, analyzing thesequence of 3'-trans-acceptor of human ACAT1 mRNA and exploring thelocalization of exon Xb in human genome. Elucidating these will benefit tofurther study of the trans-splicing mechanism. To determine whether the pre-RNAs of human ACAT1 mRNA can betrans-spliced in human cells, eukaryotic expression plasmids encoding pre-RNAsregarding as 5'-trans-donor or 3'-trans-acceptor with marker sequences wereconstructed and then transfected into human cells, trans-spliced products wasdetected by RT-PCR and DNA sequencing using exogenous marker sequences 4第一部分:人 ACAT1 mRNA 加工成熟中的反式剪接及其分子机制 摘要inserted in pre-RNAs regarding as 5'-trans-donor and 3'-trans-acceptor as primers.All the results show that exogenous pre-RNAs regarding as 5'-trans-donor or3'-trans-acceptor of human ACAT1 mRNA can be accurately trans-spliced tocorresponding endogenous or exogenous parts in human cells. To determine whether pre-RNAs of human ACAT1 mRNA can betrans-spliced in HeLa cell nuclear extract, constructs for pre-RNAs regarding as5'-trans-donor and 3'-trans-acceptor were linearized and transcribed into RNAsfor in vitro trans-splicing. RT-PCR and DNA sequencing results show thatpre-RNAs regarding as 5'-trans-donor or 3'-trans-acceptor can be accuratelytrans-spliced in HeLa cell nuclear extract. In the established in vitrotrans-splicing system, concentration and ratio of pre-RNAs regarding as5'-trans-donor or 3'-trans-acceptor, HeLa cell nuclear extract, concentration ofMg2+, incubation time, and temperature will affect the occurring and efficiency oftrans-splicing. Three concentrations (1.6 mM, 3.2 mM and 4.8 mM) of Mg2+were used to test its effect on trans-splicing, the result shows that 3.2 mM of Mg2+is the optimal for trans-splicing of human ACAT1 mRNA. The pre-RNA containing exon Xa and downstream outron (about 2 kb) canbe the trans-splicing donor, while the size of pre-RNA containing exon 1-16 (upto 100 kb) should be considerate. Thus, the role of 3'-trans-acceptor sequencefor trans-splicing of human ACAT1 mRNA was analyzed in detail. Plasmids forexpression of 3'-trans-acceptors containing serial deletions were constructed andtransfected into human cells. RT-PCR and sequencing results show that theminimal region of 3'-trans-acceptor for trans-splicing is –25 ~ +46 nt. A seriesof constructs for 3'-trans-acceptors containing introns or multiple-exons werelinearized and transcribed into RNAs. In vitro trans-splicing and RT-PCR 5第一部分:人 ACAT1 mRNA 加工成熟中的反式剪接及其分子机制 摘要results show 3'-trans-acceptors containing outron and exon 1, or with additionalpartial intron 1, or additional exon 2 present the same activity. Up to now, 10-bp CCGAATTCGG exon Xb between exon Xa (7q31) andexons 1-16 (1q25) of human ACAT1 cDNA K1 has still not been located inhuman genome. By search of published human genome sequences, 27 humangenome regions containing the 10-bp sequences were listed. Hereinto,chromosome 7 and 1 contains 1 and 4 regions respectively, which are far awayfrom downstream of Xa and upstream sequences of exon 1. There...