Expand+Functional selection and systematic analysis of intronic splicing elements identify active sequence motifs and associated splicing factors

Despite the critical role of pre-mRNA splicing in generating proteomic diversity and regulating gene expression, the sequence composition and function of intronic splicing regulatory elements (ISREs) have not been well elucidated. Here, we employed a high-throughput in vivo Screening PLatform for Intronic Control Elements (SPLICE) to identify 125 unique ISRE sequences from a random nucleotide library in human cells. Bioinformatic analyses reveal consensus motifs that resemble splicing regulatory elements and binding sites for characterized splicing factors and that are enriched in the introns of naturally occurring spliced genes, supporting their biological relevance. In vivo characterization, including an RNAi silencing study, demonstrate that ISRE sequences can exhibit combinatorial regulatory activity and that multiple trans-acting factors are involved in the regulatory effect of a single ISRE. Our work provides an initial examination into the sequence characteristics and function of ISREs, providing an important contribution to the splicing code

MBNL1 binds GC motifs embedded in pyrimidines to regulate alternative splicing

Muscleblind-like 1 (MBNL1) regulates alternative splicing and is a key player in the disease mechanism of myotonic dystrophy (DM). In DM, MBNL1 becomes sequestered to expanded CUG/CCUG repeat RNAs resulting in splicing defects, which lead to disease symptoms. In order to understand MBNL1’s role in both the disease mechanism of DM and alternative splicing regulation, we sought to identify its RNA-binding motif. A doped SELEX was performed on a known MBNL1-binding site. After five rounds of SELEX, MBNL1 selected pyrimidine-rich RNAs containing YGCY motifs. Insertion of multiple YGCY motifs into a normally MBNL1-independent splicing reporter was sufficient to promote regulation by MBNL1. MBNL1 was also shown to regulate the splicing of exon 22 in the ATP2A1 pre-mRNA, an exon mis-spliced in DM, via YGCY motifs. A search for YGCY motifs in 24 pre-mRNA transcripts that are mis-spliced in DM1 patients revealed an interesting pattern relative to the regulated exon. The intronic regions upstream of exons that are excluded in normal tissues relative to DM1, are enriched in YGCY motifs. Meanwhile, the intronic regions downstream of exons that are included in normal tissues relative to DM1, are enriched in YGCY motifs

Identification of motifs that function in the splicing of non-canonical introns

The enrichment of specific intronic splicing enhancers upstream of weak PY tracts suggests a novel mechanism for intron recognition that compensates for a weakened canonical pre-mRNA splicing motif

Modelling Silicate - Nitrate - Ammonium Co-Limitation of Algal Growth and the Importance of Bacterial Remineralisation Based on an Experimental Arctic Coastal Spring Bloom Culture Study

Arctic coastal ecosystems are rapidly changing due to climate warming, which makes modelling their productivity crucially important to better understand future changes. System primary production in these systems is highest during the pronounced spring bloom, typically dominated by diatoms. Eventually the spring blooms terminate due to silicon or nitrogen limitation. Bacteria can play an important role for extending bloom duration and total CO2 fixation through ammonium regeneration. Current ecosystem models often simplify the effects of nutrient co-limitations on algal physiology and cellular ratios and neglect bacterial driven regeneration, leading to an underestimation of primary production. Detailed biochemistry- and cell-based models can represent these dynamics but are difficult to tune in the environment. We performed a cultivation experiment that showed typical spring bloom dynamics, such as extended algal growth via bacteria ammonium remineralisation, and reduced algal growth and inhibited chlorophyll synthesis under silicate limitation, and gradually reduced nitrogen assimilation and chlorophyll synthesis under nitrogen limitation. We developed a simplified dynamic model to represent these processes. The model also highlights the importance of organic matter excretion, and post bloom ammonium accumulation. Overall, model complexity is comparable to other ecosystem models used in the Arctic while improving the representation of nutrient co-limitation related processes. Such model enhancements that now incorporate increased nutrient inputs and higher mineralization rates in a warmer climate will improve future predictions in this vulnerable system

Protein-mediated protection as the predominant mechanism for defining processed mRNA termini in land plant chloroplasts

Most chloroplast mRNAs are processed from larger precursors. Several mechanisms have been proposed to mediate these processing events, including site-specific cleavage and the stalling of exonucleases by RNA structures. A protein barrier mechanism was proposed based on analysis of the pentatricopeptide repeat (PPR) protein PPR10: PPR10 binds two intercistronic regions and impedes 5′- and 3′-exonucleases, resulting in processed RNAs with PPR10 bound at the 5′- or 3′-end. In this study, we provide evidence that protein barriers are the predominant means for defining processed mRNA termini in chloroplasts. First, we map additional RNA termini whose arrangement suggests biogenesis via a PPR10-like mechanism. Second, we show that the PPR protein HCF152 binds to the immediate 5′- or 3′-termini of transcripts that require HCF152 for their accumulation, providing evidence that HCF152 defines RNA termini by blocking exonucleases. Finally, we build on the observation that the PPR10 and HCF152 binding sites accumulate as small chloroplast RNAs to infer binding sites of other PPR proteins. We show that most processed mRNA termini are represented by small RNAs whose sequences are highly conserved. We suggest that each such small RNA is the footprint of a PPR-like protein that protects the adjacent RNA from degradation

A comprehensive computational characterization of conserved mammalian intronic sequences reveals conserved motifs associated with constitutive and alternative splicing

Orthologous mammalian introns contain many highly conserved sequences. Of these sequences, many are likely to represent protein binding sites that are under strong positive selection. In order to identify conserved protein binding sites that are important for splicing, we analyzed the composition of intronic sequences that are conserved between human and six eutherian mammals. We focused on all completely conserved sequences of seven or more nucleotides located in the regions adjacent to splice-junctions. We found that these conserved intronic sequences are enriched in specific motifs, and that many of these motifs are statistically associated with either alternative or constitutive splicing. In validation of our methods, we identified several motifs that are known to play important roles in alternative splicing. In addition, we identified several novel motifs containing GCT that are abundant and are associated with alternative splicing. Furthermore, we demonstrate that, for some of these motifs, conservation is a strong indicator of potential functionality since conserved instances are associated with alternative splicing while nonconserved instances are not. A surprising outcome of this analysis was the identification of a large number of AT-rich motifs that are strongly associated with constitutive splicing. Many of these appear to be novel and may represent conserved intronic splicing enhancers (ISEs). Together these data show that conservation provides important insights into the identification and possible roles of cis-acting intronic sequences important for alternative and constitutive splicing