title: The 0.3-kb fragment containing the R-U5-5'leader sequence of Friend murine leukemia virus influences the level of protein expression from spliced mRNA.
abstract: BACKGROUND
  A neuropathogenic variant of Friend murine leukemia virus (Fr-MLV) clone A8 induces spongiform neurodegeneration when infected into neonatal rats. Studies with chimeras constructed from the A8 virus and the non-neuropathogenic Fr-MLV clone 57 identified a 0.3-kb KpnI-AatII fragment containing a R-U5-5'leader sequence as an important determinant for inducing spongiosis, in addition to the env gene of A8 as the primary determinant. This 0.3-kb fragment contains a 17-nucleotide difference between the A8 and 57 sequences. We previously showed that the 0.3-kb fragment influences expression levels of Env protein in both cultured cells and rat brain, but the corresponding molecular mechanisms are not well understood.
  
  
  RESULTS
  Studies with expression vectors constructed from the full-length proviral genome of Fr-MLV that incorporated the luciferase (luc) gene instead of the env gene found that the vector containing the A8-0.3-kb fragment yielded a larger amount of spliced luc-mRNA and showed higher expression of luciferase when compared to the vector containing the 57-0.3-kb fragment. The amount of total transcripts from the vectors, the poly (A) tail length of their mRNAs, and the nuclear-cytoplasm distribution of luc-mRNA in transfected cells were also evaluated. The 0.3-kb fragment did not influence transcription efficiency, mRNA polyadenylation or nuclear export of luc-mRNA. Mutational analyses were carried out to determine the importance of nucleotides that differ between the A8 and 57 sequences within the 0.3-kb fragment. In particular, seven nucleotides upstream of the 5'splice site (5'ss) were found to be important in regulating the level of protein expression from spliced messages. Interestingly, these nucleotides reside within the stem-loop structure that has been speculated to limit the recognition of 5'ss.
  
  
  CONCLUSIONS
  The 0.3-kb fragment containing the R-U5-5'leader sequence of Fr-MLV influences the level of protein expression from the spliced-mRNA by regulating the splicing efficiency rather than transcription, nuclear export of spliced-mRNA, or poly (A) addition to mRNA. Seven nucleotides in the 0.3-kb fragment, which reside within the stem-loop structure that has been speculated to limit recognition of the 5'ss, could pinpoint the function of this region.
authors.affiliation: Department of Bioinformatics, Faculty of Engineering, Soka University, Hachioji, Tokyo 192-8577, Japan.
authors.forename: Yeng Cheng
authors.initials: YC
authors.lastname: Choo
authors.forename: Yohei
authors.initials: Y
authors.lastname: Seki
authors.forename: Akihito
authors.initials: A
authors.lastname: Machinaga
authors.forename: Nobuo
authors.initials: N
authors.lastname: Ogita
authors.forename: Sayaka
authors.initials: S
authors.lastname: Takase-Yoden
mesh_terms: D020121:5' Untranslated Regions; D000818:Animals; D019677:Artificial Gene Fusion; D005622:Friend murine leukemia virus / Q000235:genetics*; D015967:Gene Expression Regulation, Viral; D017930:Genes, Reporter; D008156:Luciferases / Q000032:analysis / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics*; D012367:RNA, Viral / Q000235:genetics*; D051381:Rats; D014759:Viral Envelope Proteins / Q000096:biosynthesis*
chemical_list: D020121:5' Untranslated Regions; D012333:RNA, Messenger; D012367:RNA, Viral; D014759:Viral Envelope Proteins; D008156:Luciferases