HRP-2, the Caenorhabditis elegans Homolog of Mammalian Heterogeneous Nuclear Ribonucleoproteins Q and R, Is an Alternative Splicing Factor That Binds to UCUAUC Splicing Regulatory Elements

Alternative splicing is regulated by cis sequences in the pre-mRNA that serve as binding sites for trans-acting alternative splicing factors. In a previous study, we used bioinformatics and molecular biology to identify and confirm that the intronic hexamer sequence UCUAUC is a nematode alternative splicing regulatory element. In this study, we used RNA affinity chromatography to identify trans factors that bind to this sequence. HRP-2, the Caenorhabditis elegans homolog of human heterogeneous nuclear ribonucleoproteins Q and R, binds to UCUAUC in the context of unc-52 intronic regulatory sequences as well as to RNAs containing tandem repeats of this sequence. The three Us in the hexamer are the most important determinants of this binding specificity. We demonstrate, using RNA interference, that HRP-2 regulates the alternative splicing of two genes, unc-52 and lin-10, both of which have cassette exons flanked by an intronic UCUAUC motif. We propose that HRP-2 is a protein responsible for regulating alternative splicing through binding interactions with the UCUAUC sequence.Alternative pre-mRNA splicing is a mechanism for generating multiple mRNA isoforms from a single gene. This process can allow a gene to encode for more than one protein isoform. For some genes, it is a mechanism for regulating message stability through production of alternative mRNA isoforms that are substrates for the nonsense-mediated mRNA decay pathway (1). The majority of human genes undergo alternative splicing (2), and the process can be regulated in tissue-specific and developmental stage-specific manners. Current models propose that cis elements on the pre-mRNA, in exons and introns, serve as recognition sites for trans-acting protein factors that bind to the pre-mRNA and regulate assembly of the splicing machinery, thus regulating splice site choice (3).In recent years, a number of groups have employed bioinformatics techniques to identify cis splicing regulatory elements (4). These techniques include using multiple interspecies sequence alignments to identify conserved intronic regions, identification of short sequences in exons that are bounded by weak consensus splice sites, and identification of common intronic sequences flanking similarly regulated alternative exons (5–9). These efforts have added many new sequences to the list of known and potential splicing regulators. The identification of the protein factor partners for these sequences will be important for understanding their function in alternative splicing regulation.Experimental approaches have identified alternative splicing factors that interact with specific cis elements (10), but the number of trans factors discovered still lags behind the number of newly identified cis element partners. Some examples of well-characterized cis element/trans-acting factor interactions include the NOVA K homology domain splicing factor binding to the sequence UCAY (11), the FOX splicing factors binding to the sequence UGCAUG (12–14), and hnRNP³ F/H proteins binding to the sequence GGGG (15, 16). By using cross-linking immunoprecipitation followed by large scale sequencing, entire catalogs of RNAs that the splicing factors NOVA, SF2/ASF, and FOX2 bind to in vivo have been determined (17–19). These approaches have led to models for how the proteins binding to their cis regulatory elements may alter splicing. These models include a role for the relative position of a cis element to an alternative cassette exon in determining alternative exon inclusion or skipping (18, 19).In a previous bioinformatics analysis of evolutionarily conserved intronic sequences flanking alternatively spliced exons, we identified the hexamer sequence UCUAUC as a novel splicing regulatory element (8). UCUAUC is found flanking both sides of alternative exon 16 of the unc-52 gene of Caenorhabditis elegans. Genetic analysis of a class of viable unc-52 mutants led to the discovery that exons 16–18 are alternative cassette exons and that every combination of skipping and inclusion of these three exons occurs (20). This splicing is regulated by the alternative splicing factor MEC-8 (21). Fig. 1A shows a schematic diagram of the alternatively spliced region of unc-52, with the MEC-8-enhanced alternative splicing events indicated. Using an unc-52 splicing reporter trans gene containing alternative exons 15–19, we previously reported that alternative splicing is regulated by the intronic motif UCUAUC in the intron downstream of exon 16 (8). In addition we showed that this element works cooperatively with a UGCAUG hexamer (the consensus FOX-1-binding site) in the upstream intron to regulate alternative splicing (8).Open in a separate windowFIGURE 1.RNA affinity chromatography identifies HRP-2 as binding to UCUAUC elements. A, schematic representation of the alternatively spliced region of unc-52 (adapted from Ref. 21). The alternative splicing events promoted by MEC-8 are indicated by bold lines. The lines next to introns 15 and 16 are the sites of the UCUAUC elements in those introns whose sequences were used in the RNA affinity chromatography. B, table showing sequences of RNAs immobilized to beads in the RNA affinity chromatography experiment. C, Coomassie-stained SDS-PAGE analysis of RNA affinity chromatography. C. elegans embryo extract was incubated with the different immobilized RNA substrates listed on top of the gel. Proteins identified by mass spectrometry are listed to the right of the gel, with arrows pointing to coincident protein bands. D, the left panel shows the silver stain result for the RNA affinity chromatography experiment. Each lane represents a different immobilized substrate, as indicated above. The band corresponding to HRP-2 is indicated by an arrow. The right panel is an immunoblot of the same gel using anti-HRP-2 polyclonal antibody. E, anti-HRP-2 immunoblot of an RNA affinity chromatography experiment for the indicated substrates.In this study, we report the results of a biochemical identification of a protein factor from C. elegans that binds to the UCUAUC intronic splicing regulatory element. We transcribed different short RNA sequences containing the UCUAUC element in its native intronic context, or as part of a repeating unit, and immobilized these onto agarose beads. After passing embryo extracts across these beads, we found that the protein HRP-2, the C. elegans homolog of the mammalian hnRNP Q/R proteins, binds to this sequence with high affinity. By using RNAi to reduce the level of HRP-2 in worms, we observed changes in alternative splicing of unc-52 and lin-10, two genes that contain UCUAUC elements in introns flanking alternative exons. We propose that HRP-2 is an alternative splicing factor that works through the UCUAUC intronic elements to regulate alternative splicing.