3′-End Formation of Baculovirus Late RNAs

Abstract

Baculovirus late RNAs are transcribed by a four-subunit RNA polymerase that is virus encoded. The late viral mRNAs are capped and polyadenylated, and we have previously shown that capping is mediated by the LEF-4 subunit of baculovirus RNA polymerase. Here we report studies undertaken to determine the mechanism of 3-end formation. A globin cleavage/polyadenylation signal, which was previously shown to direct 3-end formation of viral RNAs in vivo, was cloned into a baculovirus transcription template. In vitro assays with purified baculovirus RNA polymerase revealed that 3 ends were formed not by a cleavage mechanism but rather by termination after transcription of a T-rich region of the globin sequence. Terminated RNAs were released from ternary complexes and were subsequently polyadenylated. Mutational analyses indicated that the T-rich sequence was essential for termination and polyadenylation, but the poly(A) signal and the GT-rich region of the globin polyadenylation/cleavage signal were not required. Termination was not dependent on ATP hydrolysis, indicating a slippage mechanism.mRNA 3Ј-end formation is a complicated process that requires protein-nucleic acid and protein-protein interactions. The 3Ј ends of most eukaryotic RNA polymerase II transcripts are produced by a coupled cleavage/polyadenylation reaction that requires multiple factors (reviewed in references 8, 22, and 43). The processing machinery is recruited to transcription complexes by TFIID (9) and is targeted to the phosphorylated carboxy-terminal domain (CTD) of RNA polymerase II during transcription elongation (27). Recent studies have shown that RNA polymerase II itself is an essential factor in the polyadenylation step (20).Transcription termination by RNA polymerase II occurs downstream of the cleavage site and is less well understood. Termination is defined as the process of disrupting the transcription ternary complex, in which the RNA polymerases cease their elongation function, the nascent transcripts are released, and the RNA polymerases step off from the DNA templates. Disassembly of an RNA polymerase ternary complex is mediated by termination sequences that are recognized by the RNA polymerase or by termination factors (reviewed in references 37 and 39). Failure of the elongating polymerase to stop transcription before reaching an adjacent gene can lead to a reduction in the expression of the downstream gene by destabilizing the assembly of transcription initiation factors on the adjacent promoter (3,15,16,19,31). This is especially important to the gene expression programs of compact genomes, such as yeast and viruses, which have short intergenic sequences.Baculoviruses are large double-stranded DNA viruses that have been extensively used as protein expression vectors. The genomes of several baculovirus species have been sequenced and shown to be highly compressed (1,2,14). Viral gene expression is regulated in a temporal pattern (4, 11, 30) in which immediate-and delayed-early genes are transcribed by host cell RNA polymerase II,...

The Transcriptome of the Baculovirus Autographa californica Multiple Nucleopolyhedrovirus in Trichoplusia ni Cells

“…We also identified a U-rich motif located 2 to 10 nt downstream of the PAS in 58 transcripts (see Fig. S1B, panel c), consistent with observations from eukaryotic genes, but we did not frequently observe evidence of transcription termination and polyadenylation following T-rich sequences, as previously suggested (69). Only approximately 13% of late transcripts contain T-rich sequences immediately upstream of the mapped PAS.…”

“…Nevertheless, any nonpolyadenylated transcripts, should they exist, would not be detected by our methods. To better understand how transcripts are terminated and polyadenylated, we also analyzed the sequence 50 nt upstream and downstream from the PAS and found that the AT content of the 3= UTR was higher (71%) than the average AT content of the AcMNPV genome (59%), an observation consistent with prior anecdotal observations (69). The AT content in the 40 nt region immediately surrounding the PAS (Ϯ20 nt) was even higher at approximately 80% (see Fig.…”

“…The assembly and positioning of enzymatic machinery on the mRNA are then followed by endonucleolytic cleavage and subsequent addition of adenine residues by a nuclear poly(A) polymerase. In a prior study of baculovirus polyadenylation, transcripts (generated from a heterologous globin gene construct by a purified late RNA polymerase in a cell-free system) were terminated and polyadenylated by the late RNA polymerase (69). Those authors suggested that the eukaryotic polyadenylation signal motif was not required and demonstrated that the viral late RNA polymerase was capable of transcription termination and polyadenylation at the end of T-rich sequences in that in vitro system.…”

In Vitro Activity of the Baculovirus Late Expression Factor LEF-5

“…Baculovirus RNA polymerase was purified from Spodoptera frugiperda cells infected with a mixture of four viruses, each expressing one subunit of RNA polymerase, as previously described (8,16). Assays were performed using nucleoside-free cassettes linked to either the very late polyhedrin promoter pPolh/CFS or the late 39k promoter p39kL/CFS as previously described (35).…”

Functional dissection of the baculovirus late expression factor-8 gene: sequence requirements for late gene promoter activation

“…This simple four-subunit enzyme has the ability to bind DNA (Guarino et al, 1998b) and, potentially, to modify the 59 (Gross & Shuman, 1998;Guarino et al, 1998a;Jin et al, 1998) and 39 ends (Jin & Guarino, 2000) of transcripts. Although the specific role of each subunit in transcription is not known, the product of lef-4 (LEF-4) has RNA 59-triphosphatase, nucleoside triphosphatase and guanylyltransferase activities, predicting its role as a capping enzyme (Gross & Shuman, 1998;Guarino et al, 1998a;Jin et al, 1998).…”