Sequences within the 5′ Untranslated Region Regulate the Levels of a Kinetoplast DNA Topoisomerase mRNA during the Cell Cycle

Abstract

Gene expression in trypanosomatids appears to be regulated largely at the posttranscriptional level and involves maturation of mRNA precursors by trans splicing of a 39-nucleotide miniexon sequence to the 5' end of the mRNA and cleavage and polyadenylation at the 3' end of the mRNA. To initiate the identification of sequences involved in the periodic expression of DNA replication genes in trypanosomatids, we have mapped splice acceptor sites in the 5' flanking region of the TOP2 gene, which encodes the kinetoplast DNA topoisomerase, and have carried out deletion analysis of this region on a plasmid-encoded TOP2 gene. Block deletions within the 5' untranslated region (UTR) identified two regions (-608 to -388 and -387 to -186) responsible for periodic accumulation of the mRNA. Deletion of one or the other of these sequences had no effect on periodic expression of the mRNA, while deletion of both regions resulted in constitutive expression of the mRNA throughout the cell cycle. Subcloning of these sequences into the 5' UTR of a construct lacking both regions of the TOP2 5' UTR has shown that an octamer consensus sequence present in the 5' UTR of the TOP2, RPA1, and DHFR-TS mRNAs is required for normal cycling of the TOP2 mRNA. Mutation of the consensus octamer sequence in the TOP2 5' UTR in a plasmid construct containing only a single consensus octamer and that shows normal cycling of the plasmid-encoded TOP2 mRNA resulted in substantial reduction of the cycling of the mRNA level. These results imply a negative regulation of TOP2 mRNA during the cell cycle by a mechanism involving redundant elements containing one or more copies of a conserved octamer sequence within the 5' UTR of TOP2 mRNA.

Nulear Extracts of Crithidia fasciculata Contain a Factor(s) That Binds to the 5′-Untranslated Regions of TOP2 and RPA1 mRNAs Containing Sequences Required for Their Cell Cycle Regulation

“…This fragment contains part of the lacZ gene. Construction of p⌬10Not has been described previously (15).…”

“…Hydroxyurea Synchronization-The synchronization of C. fasciculata cultures by hydroxyurea was performed as described previously (4,15). Nuclear and cytosolic extracts were prepared from cultures 90 and 180 min after release from hydroxyurea arrest and assayed for binding activity.…”

“…The steady state levels of the mRNAs for these two genes and that of dihydrofolate reductase-thymidylate synthase gene are cell cycle-regulated in a similar manner (4). Sequence elements required for periodic accumulation of RPA1 and TOP2 mRNAs are present in the 5Ј-untranslated region (15,16). Examination of the 5Ј-UTR of these genes reveals the presence of a consensus octamer sequence with a conserved hexameric core.…”

Presence of Multiple mRNA Cycling Sequence Element-binding Proteins in Crithidia fasciculata

“…In synchronized cultures, the transcript levels of the genes encoding the large and middle subunits of the nuclear protein RPA (RPA1 and RPA2, a homolog of the human replication protein A), dihydrofolate reductase-thymidylate synthase (DHFR-TS), the kinetoplastspecific topoisomerase II (TOP2), and the histone-like kinetoplast-associated protein 3 have been shown to cycle in parallel, reaching maximum levels during the late G 1 and S phases and then declining rapidly during the G 2 and M phases (15). Transcripts of these genes were found to possess one or more copies of a consensus octameric sequence (C/A)AUAGAA(G/A) with a highly conserved hexameric core in either their 5Ј-or 3Ј-UTR (16,17). The sequence has been proposed to have a destabilizing role as mutations in the octamer sequence lead to accumulation of the messages to the highest level (16).…”

“…Transcripts of these genes were found to possess one or more copies of a consensus octameric sequence (C/A)AUAGAA(G/A) with a highly conserved hexameric core in either their 5Ј-or 3Ј-UTR (16,17). The sequence has been proposed to have a destabilizing role as mutations in the octamer sequence lead to accumulation of the messages to the highest level (16). Introduction of six copies of the octamer sequence (6ϫ octamer RNA) in the 5Ј-UTR of a gene that does not cycle under normal conditions resulted in cycling of the message (18).…”