Steffen Erkelenz

Human SAP18 mediates assembly of a splicing regulatory multiprotein complex via its ubiquitin-like fold

RNPS1, Acinus, and SAP18 form the apoptosis- and splicing-associated protein (ASAP) complex, which is also part of the exon junction complex. Whereas RNPS1 was originally identified as a general activator of mRNA processing, all three proteins have been found within functional spliceosomes. Both RNPS1 and Acinus contain typical motifs of splicing regulatory proteins including arginine/serine-rich domains. Due to the absence of such structural features, however, a function of SAP18 in splicing regulation is completely unknown. Here we have investigated splicing regulatory activities of the ASAP components. Whereas a full-length Acinus isoform displayed only limited splicing regulatory activity, both RNPS1 and, surprisingly, SAP18 strongly modulated splicing regulation. Detailed mutational analysis and three-dimensional modeling data revealed that the ubiquitin-like fold of SAP18 was required for efficient splicing regulatory activity. Coimmunoprecipitation and immunofluorescence experiments demonstrated that SAP18 assembles a nuclear speckle-localized splicing regulatory multiprotein complex including RNPS1 and Acinus via its ubiquitin-like fold. Our results therefore suggest a novel function of SAP18 in splicing regulation.

Genomic HEXploring allows landscaping of novel potential splicing regulatory elements

Effective splice site selection is critically controlled by flanking splicing regulatory elements (SREs) that can enhance or repress splice site use. Although several computational algorithms currently identify a multitude of potential SRE motifs, their predictive power with respect to mutation effects is limited. Following a RESCUE-type approach, we defined a hexamer-based ‘HEXplorer score’ as average Z-score of all six hexamers overlapping with a given nucleotide in an arbitrary genomic sequence. Plotted along genomic regions, HEXplorer score profiles varied slowly in the vicinity of splice sites. They reflected the respective splice enhancing and silencing properties of splice site neighborhoods beyond the identification of single dedicated SRE motifs. In particular, HEXplorer score differences between mutant and reference sequences faithfully represented exonic mutation effects on splice site usage. Using the HIV-1 pre-mRNA as a model system highly dependent on SREs, we found an excellent correlation in 29 mutations between splicing activity and HEXplorer score. We successfully predicted and confirmed five novel SREs and optimized mutations inactivating a known silencer. The HEXplorer score allowed landscaping of splicing regulatory regions, provided a quantitative measure of mutation effects on splice enhancing and silencing properties and permitted calculation of the mutationally most effective nucleotide.

An intronic G-run within HIV-1 intron 2 is critical for splicing regulation of vif-mRNA

ABSTRACT Within target T lymphocytes, human immunodeficiency virus type I (HIV-1) encounters the retroviral restriction factor APOBEC3G (apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like 3G; A3G), which is counteracted by the HIV-1 accessory protein Vif. Vif is encoded by intron-containing viral RNAs that are generated by splicing at 3′ splice site (3′ss) A1 but lack splicing at 5′ss D2, which results in the retention of a large downstream intron. Hence, the extents of activation of 3′ss A1 and repression of D2, respectively, determine the levels of vif mRNA and thus the ability to evade A3G-mediated antiviral effects. The use of 3′ss A1 can be enhanced or repressed by splicing regulatory elements that control the recognition of downstream 5′ss D2. Here we show that an intronic G run (GI2-1) represses the use of a second 5′ss, termed D2b, that is embedded within intron 2 and, as determined by RNA deep-sequencing analysis, is normally inefficiently used. Mutations of GI2-1 and activation of D2b led to the generation of transcripts coding for Gp41 and Rev protein isoforms but primarily led to considerable upregulation of vif mRNA expression. We further demonstrate, however, that higher levels of Vif protein are actually detrimental to viral replication in A3G-expressing T cell lines but not in A3G-deficient cells. These observations suggest that an appropriate ratio of Vif-to-A3G protein levels is required for optimal virus replication and that part of Vif level regulation is effected by the novel G run identified here.

Limited complementarity between U1 snRNA and a retroviral 5' splice site permits its attenuation via RNA secondary structure

Multiple types of regulation are used by cells and viruses to control alternative splicing. In murine leukemia virus, accessibility of the 5′ splice site (ss) is regulated by an upstream region, which can fold into a complex RNA stem–loop structure. The underlying sequence of the structure itself is negligible, since most of it could be functionally replaced by a simple heterologous RNA stem–loop preserving the wild-type splicing pattern. Increasing the RNA duplex formation between U1 snRNA and the 5′ss by a compensatory mutation in position +6 led to enhanced splicing. Interestingly, this mutation affects splicing only in the context of the secondary structure, arguing for a dynamic interplay between structure and primary 5′ss sequence. The reduced 5′ss accessibility could also be counteracted by recruiting a splicing enhancer domain via a modified MS2 phage coat protein to a single binding site at the tip of the simple RNA stem–loop. The mechanism of 5′ss attenuation was revealed using hyperstable U1 snRNA mutants, showing that restricted U1 snRNP access is the cause of retroviral alternative splicing.

Tra2-Mediated Recognition of HIV-1 5′ Splice Site D3 as a Key Factor in the Processing of vpr mRNA

ABSTRACT Small noncoding HIV-1 leader exon 3 is defined by its splice sites A2 and D3. While 3′ splice site (3′ss) A2 needs to be activated for vpr mRNA formation, the location of the vpr start codon within downstream intron 3 requires silencing of splicing at 5′ss D3. Here we show that the inclusion of both HIV-1 exon 3 and vpr mRNA processing is promoted by an exonic splicing enhancer (ESE vpr ) localized between exonic splicing silencer ESSV and 5′ss D3. The ESE vpr sequence was found to be bound by members of the Transformer 2 (Tra2) protein family. Coexpression of these proteins in provirus-transfected cells led to an increase in the levels of exon 3 inclusion, confirming that they act through ESE vpr . Further analyses revealed that ESE vpr supports the binding of U1 snRNA at 5′ss D3, allowing bridging interactions across the upstream exon with 3′ss A2. In line with this, an increase or decrease in the complementarity of 5′ss D3 to the 5′ end of U1 snRNA was accompanied by a higher or lower vpr expression level. Activation of 3′ss A2 through the proposed bridging interactions, however, was not dependent on the splicing competence of 5′ss D3 because rendering it splicing defective but still competent for efficient U1 snRNA binding maintained the enhancing function of D3. Therefore, we propose that splicing at 3′ss A2 occurs temporally between the binding of U1 snRNA and splicing at D3.

Balanced splicing at the Tat-specific HIV-1 3'ss A3 is critical for HIV-1 replication

BackgroundThe viral regulatory protein Tat is essential for establishing a productive transcription from the 5′-LTR promoter during the early phase of viral gene expression. Formation of the Tat-encoding mRNAs requires splicing at the viral 3′ss A3, which has previously been shown to be both negatively and positively regulated by the downstream splicing regulatory elements (SREs) ESS2p and ESE2/ESS2. However, using the novel RESCUE-type computational HEXplorer algorithm, we were recently able to identify another splicing enhancer (ESE5807-5838, henceforth referred to as ESEtat) located between ESS2p and ESE2/ESS2. Here we show that ESEtat has a great impact on viral tat-mRNA splicing and that it is fundamental for regulated 3′ss A3 usage.ResultsMutational inactivation or locked nucleic acid (LNA)-directed masking of the ESEtat sequence in the context of a replication-competent virus was associated with a failure (i) to activate viral 3′ss A3 and (ii) to accumulate Tat-encoding mRNA species. Consequently, due to insufficient amounts of Tat protein efficient viral replication was drastically impaired. RNA in vitro binding assays revealed SRSF2 and SRSF6 as candidate splicing factors acting through ESEtat and ESE2 for 3′ss A3 activation. This notion was supported by coexpression experiments, in which wild-type, but not ESEtat-negative provirus responded to higher levels of SRSF2 and SRSF6 proteins with higher levels of tat-mRNA splicing. Remarkably, we could also find that SRSF6 overexpression established an antiviral state within provirus-transfected cells, efficiently blocking virus particle production. For the anti-HIV-1 activity the arginine-serine (RS)-rich domain of the splicing factor was dispensable.ConclusionsBased on our results, we propose that splicing at 3′ss A3 is dependent on binding of the enhancing SR proteins SRSF2 and SRSF6 to the ESEtat and ESE2 sequence. Mutational inactivation or interference specifically with ESEtat activity by LNA-directed masking seem to account for an early stage defect in viral gene expression, probably by cutting off the supply line of Tat that HIV needs to efficiently transcribe its genome.

A natural tapasin isoform lacking exon 3 modifies peptide loading complex function

To assure efficient MHC class I (MHC‐I) peptide loading, the peptide loading complex (PLC) recruits the peptide‐receptive form of MHC‐I, and in this process, tapasin (tpn) connects MHC‐I with the peptide transporter TAP and forms a stable disulfide bond with ERp57. Here, we describe an alternatively spliced tpn transcript lacking exon 3, observed in cells infected with human cytomegalovirus. Recognition of exon 3 was regulated via G‐runs, suggesting that members of the hnRNP (heterogeneous nuclear ribonucleoprotein)‐family regulate expression of the ΔExon3 variant of tpn. Exon 3 includes Cys‐95, which is responsible for the disulfide bond formation with ERp57 and, consequently, interaction of the ΔExon3 variant with ERp57 was strongly impaired. Although the ΔExon3 variant specifically stabilized TAP expression but not MHC‐I in tpn‐deficient cells, in tpn‐proficient cells, the ΔExon3 tpn reduced cell surface expression of the tpn‐dependent HLA‐B*44:02 allele; the stability of the tpn‐independent HLA‐B*44:05 was not affected. Most importantly, detailed analysis of the PLC revealed a simultaneous binding of the ΔExon3 variant and tpn to TAP, suggesting modification of PLC functions. Indeed, an altered MHC‐I ligandome was observed in HeLa cells overexpressing the ΔExon3 variant, highlighting the potential of the alternatively spliced tpn variant to impact CD8+ T‐cell responses.

Frequent Gain and Loss of Intronic Splicing Regulatory Elements during the Evolution of Vertebrates

Splicing regulatory elements (SREs) are sequences bound by proteins that influence splicing of nearby splice sites. Constitutively spliced introns have evolved to utilize many different splicing factors. The evolutionary processes that influenced which splicing factors are used for splicing of individual introns are generally unclear. We demonstrate that in the lineage that gave rise to mammals, many introns lost U-rich sequences and gained G-rich sequences, both of which resemble known SREs. The apparent conversion of U-rich to G-rich SREs suggests that the associated splicing factors are functionally equivalent. In support of this we demonstrated that U-rich and G-rich SREs are both capable of promoting splicing of an SRE-dependent splicing reporter. Furthermore, we demonstrate, using the heterologous MS2 tethering system (bacterial MS2 coat fusion-protein and its RNA stem-loop binding site), that both the U-rich SRE-binding protein (TIA1) and the G-rich SRE-binding protein (HNRNPF) can promote splicing of the same intron. We also observed that gain of G-rich SREs is significantly associated with G/C-rich genomic isochores, suggesting that gain or loss of SREs was driven by the same processes that ultimately resulted in the formation of mammalian genomic isochores. We propose the following model for the gain and loss of mammalian SREs. Ancestral U-rich SREs located in genomic regions that were experiencing high rates of A/T to G/C conversion would have suffered frequent deleterious mutations. However, this same process resulted in increased formation of functionally equivalent G-rich SREs, and acquisition of new G-rich SREs decreased purifying selection on the U-rich SREs, which were then free to decay.

The PI3K pathway acting on alternative HIV-1 pre-mRNA splicing.

HIV-1 mediates pro-survival signals and prevents apoptosis via the phosphatidylinositol-3-kinase (PI3K) pathway. This pathway, however, also affects phosphorylation of serine-arginine (SR) proteins, a family of splicing regulatory factors balancing splice site selection. We now show that pharmacologic inhibition of PI3K signalling alters the HIV-1 splicing pattern of both minigene- and provirus-derived mRNAs. This indicates that HIV-1 might also promote PI3K signalling to balance processing of its transcripts by regulating phosphorylation of splicing regulatory proteins.

Mutational analysis of the internal membrane proximal domain of the HIV glycoprotein C-terminus.

This study focuses on the long stretch of highly conserved amino acids in the membrane proximal part of the HIV-1 cytoplasmic tail (Env amino acids (aa) 706-718) upstream of the overlap with the tat and rev second coding exons. Changes in Env aa 713 and 715, although they did not affect Env function, abrogated replicative spread. Other amino acid substitutions, i.e., 706-712, 714 and 716, despite their conservation, did not result in defective replicative phenotypes even in primary peripheral blood lymphocytes. Our results point to their involvement in presently unrecognized essential Env functions pertinent only in in vivo. Interestingly, changes in the codons for residues 717-718 as well as some mutations in residues 714-716 abrogated Gag expression but still allowed expression of functional Env in a rev-independent manner. This could be due to the inactivation of a rev-regulated negative element within the respective nucleotide sequence (8354-8368).