Precursor RNAs harboring nonsense codons accumulate near the site of transcription.

Messenger RNAs containing premature termination codons (PTCs) are selectively eliminated by nonsense-mediated mRNA decay (NMD). Paradoxically, although cytoplasmic ribosomes are the only known species capable of PTC recognition, in mammals many PTC-containing mRNAs are apparently eliminated prior to release from the nucleus. To determine whether PTCs can influence events within the nucleus proper, we studied the immunoglobulin (Ig)-mu and T cell receptor (TCR)-beta genes using fluorescent in situ hybridization (FISH). Alleles containing PTCs, but not those containing a missense mutation or a frameshift followed by frame-correcting mutations, exhibited elevated levels of pre-mRNA, which accumulated at or near the site of transcription. Our data indicate that mRNA reading frame can influence events at or near the site of gene transcription.

A novel type of splicing enhancer regulating adenovirus pre-mRNA splicing.

Splicing of the adenovirus IIIa pre-mRNA is subjected to a temporal regulation, such that efficient IIIa 3' splice site usage is confined to the late phase of the infectious cycle. Here we show that IIIa pre-mRNA splicing is activated more than 200-fold in nuclear extracts prepared from late adenovirus-infected cells (Ad-NE) compared to uninfected HeLa cell nuclear extracts (HeLa-NE). In contrast, splicing of the beta-globin pre-mRNA is repressed in Ad-NE. We constructed hybrid pre-mRNAs between IIIa and beta-globin in order to identify the minimal IIIa sequence element conferring enhanced splicing in Ad-NE. Using this approach, we show that the IIIa branch site/pyrimidine tract functions as a Janus element: it blocks splicing in HeLa-NE and functions as a splicing enhancer in Ad-NE. Therefore, we named this sequence the IIIa virus infection-dependent splicing enhancer (3VDE). This element is essential for regulated IIIa pre-mRNA splicing in Ad-NE and sufficient to confer an enhanced splicing phenotype to the beta-globin pre-mRNA in Ad-NE. We further show that the increase in IIIa splicing observed in Ad-NE is not accompanied by a similar increase in U2AF binding to the IIIa pyrimidine tract. This finding suggests that splicing activation by the 3VDE may operate without efficient U2AF interaction with the pre-mRNA. Importantly, this report represents the first description of a splicing enhancer that has evolved to function selectively in the context of a virus infection, a finding that adds a new level at which viruses may subvert the host cell RNA biosynthetic machinery to facilitate their own replication.

Regulation of adenovirus alternative RNA splicing by dephosphorylation of SR proteins.

SR proteins are a family of essential splicing factors required for early recognition of splice sites during spliceosome assembly. They also function as alternative RNA splicing factors when overexpressed in vivo or added in excess to extracts in vitro. SR proteins are highly phosphorylated in vivo, a modification that is required for their function in spliceosome assembly and splicing catalysis. Here we show that SR proteins purified from late adenovirus-infected cells are inactivated as splicing enhancer or splicing repressor proteins by virus-induced dephosphorylation. We further show that the virus-encoded protein E4-ORF4 activates dephosphorylation by protein phosphatase 2A of HeLa SR proteins and converts their splicing properties into that of SR proteins purified from late adenovirus-infected cells. Taken together, our results suggest that E4-ORF4 is an important factor controlling the temporal shift in adenovirus alternative RNA splicing. We conclude that alternative pre-mRNA splicing, like many other biological processes, is regulated by reversible protein phosphorylation.

Inhibition by SR proteins of splicing of a regulated adenovirus pre-mRNA.

The adenovirus L1 unit represents an example of an alternatively spliced precursor messenger (pre-mRNA) where on 5' splice can be jointed to one of two alternative 3' splice sites, producing the 52,55K or the IIIa mRNAs (Fig. 1a). Efficient usage of the distal IIIa 3' splice site requires late viral protein synthesis and is therefore confined to the late phase of virus infection. Here we show that, in extracts from uninfected cells, the classical SR proteins, which are essential splicing factors, inhibit IIIa pre-mRNA splicing by binding to an intronic repressor element and preventing recruitment of the U2 small nuclear ribonucleoprotein particle to the spliceosome. We further show that the viral repressor element has splicing-enhancer activity when appropriately placed in the pre-mRNA. Together, our results demonstrate that SR proteins function as activators or repressors of splicing depending on where on the pre-mRNA they bind.

Enhanced splicing of nonconsensus 3' splice sites late during adenovirus infection.

The adenovirus major late transcription unit is an example of an alternatively spliced gene, in which a common 5' splice site can be spliced to more than 15 different 3' splice sites. Here we show that the specificity in 3' splice site recognition changes during virus infection, such that 3' splice sites with long consensus-type polypyrimidine tracts are repressed while 3' splice sites with short atypical polypyrimidine tracts, which bind U2AF65K inefficiently, are enhanced in splicing in late virus-infected nuclear extracts. On the basis of these experiments, we discuss a mechanism that helps to explain how the complex pattern of major late mRNAs is produced late during virus infection.

Splicing of two weak 3' splice sites from the adenovirus major late region is enhanced in nuclear extracts from adenovirus infected cells.

The adenovirus major late transcription unit (MLTU) is an example of a complex alternatively spliced gene, in which more than 15 different 3' splice sites can be joined to a common 5' splice site. Maturation of the full repertoire of possible mRNAs requires late viral protein synthesis and occurs only at late stages of the infectious cycle (16-24 hpi). We are trying to decipher the mechanisms regulating alternative 3' splice site choice during the infectious cycle. Therefore, we examined the splicing activity of several 3' splice sites from the MLTU in vitro in nuclear extracts prepared from adenovirus infected cells (Ad NE) and from uninfected cells. The results suggest that pre-mRNAs with "weak" 3' splice sites (short, atypical polypyrimidine tracts) are activated and pre-mRNAs with long, prototypical polypyrimidine tracts are repressed in Ad NE. In fact, our data show a reciprocal correlation between the strength of a polypyrimidine tract, defined by its affinity for U2AF65K in vitro, and the efficiency of splicing in Ad NE. We are currently investigating the possible mechanisms responsible for this observed shift in 3' splice site choice during an adenovirus infection.