UvA-DARE (Digital Academic Repository) Regulation of HIV-1 splicing Müller, N. Publication date 2016 Document Version Final published version Link to publication Citation for published version (APA): Müller, N. (2016). Regulation of HIV-1 splicing. General rights It is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), other than for strictly personal, individual use, unless the work is under an open content license (like Creative Commons). Disclaimer/Complaints regulations If you believe that digital publication of certain material infringes any of your rights or (privacy) interests, please let the Library know, stating your reasons. In case of a legitimate complaint, the Library will make the material inaccessible and/or remove it from the website. Please Ask the Library: https://uba.uva.nl/en/contact, or a letter to: Library of the University of Amsterdam, Secretariat, Singel 425, 1012 WP Amsterdam, The Netherlands. You will be contacted as soon as possible. UvA-DARE is a service provided by the library of the University of Amsterdam (https://dare.uva.nl) Download date:01 Oct 2021 Chapter 6 A search for cellular 5’ splice sites that are regulated by RNA structure Nancy Muellera, Alizée Borscheb, Stephan Theissc, Heiner Schaalb, Ben Berkhouta and Atze T. Dasa* a Laboratory of Experimental Virology, Department of Medical Microbiology, Center for Infection and Immunity Amsterdam (CINIMA), Academic Medical Center, University of Amsterdam, The Netherlands b Institute for Virology, Heinrich-Heine-University Duesseldorf, Duesseldorf, Germany c Institute of Clinical Neuroscience and Medical Psychology, Heinrich-Heine- University Duesseldorf, Duesseldorf, Germany Manuscript in preparation Abstract Splicing of RNAs produced upon transcription of cellular genes results in the removal of the intronic sequences that are positioned between 5’ and 3’ splice sites. Previous studies demonstrated that the frequency of splicing at these splice sites can be influenced by local RNA structure, possibly by controlling the accessibility for splice factors. To identify cellular 5’ splice sites (5’ss) in the human transcriptome that are controlled by RNA structure, we screened a large set of 5’ss sequences from the ENSEMBL database for the capacity to fold a stable RNA structure. This resulted in the identification of multiple 5’ss-containing sequences that can fold into a hairpin structure in the primary transcript. Selected 5’ss hairpin sequences and destabilized mutants were inserted into reporter transcripts for testing of the 5’ss activity. We thus demonstrate that the 5’ss#8 site in the primary FAM73B transcript is presented in a stable hairpin structure that can inhibit splicing. Despite this inhibitory effect of the RNA structure, splicing at the 5’ss#8 was found to be complete when tested in FAM73B exon-intron containing reporter transcripts. In this more natural context, splicing was reduced by mutations that further stabilize the hairpin. These results indicate that other factors, like surrounding exon or intron sequences or the 3’ss strength, may influence the splicing frequency. In addition, we identified an alternative 5’ss in the hairpin sequence that can be activated by inactivation of 5’ss#8. Introduction Splicing is a co-transcriptional process in which intronic sequences are removed from the primary RNA transcript. The intron sequences are identified by a splice donor site (5’ splice site; 5’ss) at the 5’ side and a splice acceptor site (3’ss) at the 3’ side. The presence of alternative 5’ss and 3’ss allows the production of different mRNAs, which contributes to the proteome complexity (1). Several mutations in the human genome have been identified that affect splice site usage and cause diseases, including cancer and neurological disorders (2-12). The spliceosome complex that is formed on the primary RNA transcript consists of several small nuclear ribonucleoproteins (snRNPs). Initially, the sequence complementarity between the 5’ terminal nucleotides (nts) of U1 snRNA and the 5’ss mediates U1 snRNP binding (Fig. 1A). The U2 snRNP auxiliary factor (U2AF) interacts with the 3’ss (13, 14), followed by binding of U2 snRNP to the branch point sequence (14, 15). Subsequently, the U4/U6-U5 tri-snRNP particle will bind to complete the process of spliceosome assembly (14). The splicing efficiency at the 5’ss is controlled by different factors. Firstly, the sequence of the 5’ss influences how efficient the U1 snRNA can anneal. The hydrogen bond score (HBS; (16)) reflects the base pairing potential between U1 snRNA and 11 nts of the 5’ss (3 exonic and 8 intronic nts). The HBS value can vary from 1.8 (only the critical G+1U positions pair with U1 snRNA) to 23.8 (complete complementarity). Secondly, the splicing process can be stimulated or inhibited by splicing regulatory proteins, like SR proteins and hnRNPs, that bind to splicing regulatory elements (SREs) in close proximity to the 5’ss (17). Thirdly, the splicing frequency can be influenced by local RNA structure at the 5’ss, which can restrict access of the splicing factors, including U1 snRNP and regulatory proteins (18-30). 6 The primary HIV-1 transcript contains several 5’ss and 3’ss sites and differential use of these sites allows the production of a large variety of RNAs. These RNAs are used either as genomic RNA and packaged into new virus particles or as mRNA for the translation of all viral proteins. The balanced production of these differentially spliced RNAs is essential for efficient virus replication, which makes HIV-1 splicing an interesting model to study splicing regulation. The major 5’ss that is present in the 5’ untranslated leader of the viral transcripts is used for the production of all spliced mRNAs. Studies that investigated the regulation of splicing at this 5’ss indicated that all afore-mentioned factors, 5’ss/U1 snRNA complementarity, SRE sites and local RNA structure are involved in splicing control (31-35). The splice donor (SD) region surrounding the major 5’ss can fold a stable RNA hairpin structure that partially occludes the U1 snRNA binding site. Stabilization of this SD hairpin was found to reduce splicing, whereas destabilization of this structure increases splicing (32, 34). These results demonstrated that splicing at the major 5’ss is modulated by the stability of the SD hairpin, most likely by controlling the accessibility for splicing factors. In addition, it was shown that SR proteins bound in the SD region influence splicing and that suboptimal sequence complementarity between 5’ss and U1 snRNA prevents complete splicing (31, 35). 109 In this study, we set out to identify 5’ss sites in the human transcriptome that are controlled by local RNA structure in a similar way as the HIV-1 major 5’ss. A large set (47,279) of 5’ss-containing sequences was extracted from the ENSEMBL human genome database and analyzed for the capacity to fold a stable RNA structure. This resulted in the identification of multiple cellular 5’ss sites that adopt a stem-loop structure in the primary transcript. For several of these sites, we tested whether the splicing frequency is controlled by RNA structure. Results Selection of candidate 5’ss hairpin structures A large set (47,279) of human 5’ss sequences was extracted from the ENSEMBL human genome database (http://www.ensembl.org/index.html). To identify 5’ss sequences that are potentially masked in a local RNA structure, the secondary structure of the 5’ss region, including the 11-nt U1 snRNA binding site and 10 upstream and downstream nts (Fig. 1A) was predicted with the MFold RNA structure analysis software. The thermodynamic stability (ΔG in kcal/mol) of the “Top 100” most stable RNA structures varied from -24.7 to -15.2 kcal/mol (Supplementary Table 1). The hydrogen bond score (HBS; (16)) that reflects the base pairing potential with U1 snRNA varied from 4.8 to 19.6, with a high value reflecting significant U1 snRNA-5’ss sequence complementarity. Several 5’ss were selected for subsequent splicing analysis based on the following criteria. First, the 5’ss region should fold a hairpin that includes the complete 11-nt U1 snRNA binding site. Second, the same structure should be predicted when a larger RNA segment, including 30 nts upstream and downstream of the 11-nt 5’ss 6 sequence, is analyzed with MFold (Fig. 1A). Third, to select stable hairpin structures, the maximal loop size was arbitrarily set at 6 nts and the stem region should maximally contain a single destabilizing bulge. Fourth, the 5’ss region should not contain any AUG triplets that may interfere with proper translation of the luciferase reporter used for measuring the splicing efficiency (see below). We thus selected five 5’ss sequences that can fold a stable hairpin structure (HS1 to HS5 in Fig. 1B and C). The HBS value of these sites varied from 10.7 to 17.4 (Table 1). The selected 5’ss sequences are derived from different genes and all are constitutively used during processing of the primary transcript. Destabilization of 5’ss hairpins can increase the splicing efficiency To measure the splicing activity of the selected 5’ss sites, we made pGL3-based promoter-reporter constructs in which the 5’ss hairpin sequences were inserted in the 5’ untranslated leader of the luciferase-encoding transcript (32) (Fig. 2A). Splicing from the introduced 5’ss to a 3’ss in the luciferase sequences will yield a transcript that does not encode luciferase (Fig. 2A). To analyze the potentially suppressive effect of the 5’ss hairpin structure on splicing efficiency, the structure was destabilized by mutation of the stem sequence (HS1d-HS5d in Fig.