The coding sequences of many eukaryotic genes are interrupted by noncoding introns, which are also present in the primary transcripts, or pre-mRNAs. The introns must be precisely removed, and the coding exons joined, to allow translation of functional proteins. Pre-mRNA splicing, a nuclear process, can be constitutive or alternative. Constitutive splicing is the removal of introns by joining together all the adjacent exons in the order of their arrangement. In constitutive splicing, a single protein is produced from a single pre-mRNA, regardless of where and when the gene is expressed. In alternative splicing, variable use of splice sites allows two or more mature mRNAs to be generated from the same pre-mRNA. For example, an entire exon or part of an exon can be included or skipped in different spliced mRNAs. Alternative splicing is a prevalent way by which many eukaryotes diversify the number of proteins produced from a single pre-mRNA transcript (57
Analysis of the human genome indicated that more than 74% of human genes encode at least two isoforms by alternative splicing (27
). An extreme example of alternative splicing is the Drosophila
Dscam gene, in which a single pre-mRNA transcript apparently encodes 38,016 protein isoforms through combinatorial alternative splicing events (21
). Alternative splicing can in many cases be subject to regulation, for example, in a cell-type-specific manner, during embryonic development, or in response to signaling pathways.
Retroviruses such as human immunodeficiency virus type 1 (HIV-1) also depend on alternative splicing to produce all of the viral proteins from a single primary transcript (59
). The unspliced transcript is necessary for viral replication, packaging into virions, and translation of several proteins, whereas other viral proteins are generated from partially spliced or fully spliced transcripts. Special mechanisms allow these incompletely spliced transcripts to be exported to the cytoplasm for translation (13
Heterogeneous nuclear ribonucleoproteins (hnRNPs) are trans
-acting factors that bind to exonic splicing silencers (ESSs) or intronic splicing silencers, usually to inhibit the use of particular splice sites during regulated splicing events. There are some instances in which hnRNPs promote splicing instead of inhibiting it (8
). The most common feature of hnRNPs is the presence of two or more RNA binding domains and an auxiliary domain believed to be responsible for protein-protein, RNA-protein, and single-stranded DNA-protein interactions. Most of these hnRNPs can also form homophilic interactions and heterophilic interactions with other hnRNPs (9
). One of the most abundant hnRNPs is hnRNP A1 (17
). hnRNP A1 has been implicated in many alternative splicing events in human and several other eukaryotes (1
). Human hnRNP A1 is a 320-amino-acid protein, of which the 196-amino-acid N-terminal domain comprises two RNA recognition motifs (RRMs) (39
). The 124-amino-acid C-terminal domain is glycine rich and is believed to be responsible for cooperative binding, leading to repression of splicing (16
). At present, there are no available structures of intact hnRNP A1, but there are high-resolution crystal structures of its N-terminal domain spanning RRM1 and RRM2, which is known as unwinding protein 1 (UP1) (16
The manner in which hnRNP A1 controls alternative splicing is still not fully understood. A study from our lab focusing on splicing of exon 3 of the HIV-1 tat pre-mRNA showed an antagonistic effect of an ESS element, ESS3, mediated by hnRNP A1, vis-à-vis another cis
-acting splicing regulatory element, known as an exonic splicing enhancer (ESE) (67
). ESEs enhance splicing or promote inclusion of a particular exon through the binding of one or more activator proteins, such as members of the serine/arginine-rich (SR) family, which in turn recruit other components of the splicing machinery to the 5′ and 3′ splice sites (26
). SR proteins have one or two RRMs at their N terminus, which interact with the RNA (3
). The C-terminal domain of each SR protein comprises a highly conserved arginine/serine-rich (RS) domain; however, this domain is not always necessary for splicing (56
). SR proteins are important for the recognition of splice sites and act at the earliest stages of spliceosome assembly, as well as at later stages of splicing (10
). SR proteins have other functions in splicing and gene expression besides binding to ESEs, and they are essential for constitutive splicing (26
). Even in the case of introns with strong splice sites, in which an ESE might not be required, SR proteins are essential for recognition of the splice sites and recruitment of the splicing machinery (18
Initial high-affinity binding of hnRNP A1 to ESS3 is followed by its cooperative spreading along tat exon 3, which allows hnRNP A1 to displace the SR protein SC35 from its cognate ESE, thereby preventing splicing of tat exon 3 (67
). That same study also showed that when another SR protein, SF2/ASF, binds to its cognate ESE, hnRNP A1 cannot effectively displace it, and therefore, there is inclusion of tat exon 3 (67
). The net effect depends in part on the strength of the SR protein interaction with its cognate ESE and presumably on the nuclear abundance of particular SR proteins and hnRNP A1 in a given cell type.
There is increased expression of hnRNP A1 or SR proteins in some tumors and tumor cell lines compared to in normal cells and tissues (20
). Putting all this information together presents a strong case for studying how cooperative binding of hnRNP A1 leads to alternative splicing of a specific exon. Understanding cooperative binding of hnRNP A1 in the context of HIV tat and other model substrates is expected to shed light on the mechanisms of alternative splicing in general.
The present study addresses the mechanism of hnRNP A1 cooperative binding to RNA. We show that hnRNP A1 cooperative binding results in unwinding of RNA secondary structure. After binding to a high-affinity site, hnRNP A1 spreads preferentially, though not exclusively, in a 3′-to-5′ direction and can displace other bound proteins from the RNA to repress splicing.