SAM homeostasis is regulated by CFIm-mediated splicing of MAT2A

SUMMARYS-adenosylmethionine (SAM) is the methyl donor for nearly all cellular methylation events. Cells regulate intracellular SAM levels through intron detention of the MAT2A RNA, which encodes only SAM synthetase expressed in most cells. The N6-adenosine methyltransferase METTL16 promotes splicing of the MAT2A detained intron by an unknown mechanism. Using an unbiased CRISPR knock-out screen, we identified CFIm25 (NUDT21) to be a regulator of MAT2A intron detention and intracellular SAM levels. CFIm25 is a component of the cleavage factor Im (CFIm) complex that regulates poly(A) site selection, but we show it promotes MAT2A splicing, independent of poly(A) site selection. CFIm25-mediated MAT2A splicing induction requires the RS domains of its binding partners, CFIm68 and CFIm59 as well as binding sites in detained intron and 3′ UTR. These studies uncover mechanisms that regulate MAT2A intron detention and reveal previously undescribed roles for CFIm in splicing and SAM metabolism.

Tra2 protein biology and mechanisms of splicing control

Tra2 proteins regulate pre-mRNA splicing in vertebrates and invertebrates, and are involved in important processes ranging from brain development in mice to sex determination in fruitflies. In structure Tra2 proteins contain two RS domains (domains enriched in arginine and serine residues) flanking a central RRM (RNA recognition motif). Understanding the mechanisms of how Tra2 proteins work to control splicing is one of the key requirements to understand their biology. In the present article, we review what is known about how Tra2 proteins regulate splicing decisions in mammals and fruitflies.

Functional Analysis of SRSF2 Mutations in Myelodysplastic Syndromes and Related Disorders

Abstract 1706 The recent study of whole-exome sequencing on MDS has revealed frequent and specific pathway mutations involving multiple components of the RNA splicing machinery, including U2AF35, SRSF2, SF3B1 and ZRSR2. The mutually exclusive manner of these mutations among MDS cases also supported that deregulated RNA splicing contributes to the pathogenesis of MDS. Interestingly, the distribution of these splicing pathway mutations shows a substantial difference with regard to disease subtypes. Thus, the SF3B1 mutations are by far the most frequent in RARS and RCMD-RS cases, and the SRSF2 mutations are more prevalent in CMML. SRSF2 is a member of the SR protein family that is commonly characterized by one or two RNA recognition motifs (RRM) and a signature serine/arginine-rich domains (RS domains). The SR proteins interact with other spliceosome components through their RS domains, among which most extensively characterized are SRSF1 (ASF/SF2) and SRSF2 (SC35). Both SR proteins bind a splicing enhancer site within the 3' target exon and also interact with the U2AF, playing an indispensable role in both constitutive and alternative splicing in most cell types. In fact, the knockout of these genes in mice results in embryonic lethality. There is emerging evidence that establishes a connection between the abnormal expression of SR proteins and the development of cancer, mainly as a result of change in the alternative splicing patterns of key transcripts. Increased expression of SR proteins usually correlates with cancer progression, as shown by elevated expression of SR proteins in ovarian cancer and breast cancer. In spite of the similarity in their functions, both proteins are thought to have distinct roles, especially in the pathogenesis of myeloid malignancies, since we found no SRSF1 mutations among 582 cases with myeloid neoplasms. On the other hand, studies have shown that increased expression of SRSF1 transforms immortal rodent fibroblasts and leads to the formation of sarcomas in nude mice, supporting the notion that SRSF1 is a proto-oncogene, whereas SRSF2 does not have transforming activity, indicating a highly specific role of SRSF1 in this type of cancer. Thus, little is known about the biological mechanism by which the SRSF2 mutations are involved in the pathogenesis of MDS, although the mutations at the P95 site are predicted to cause a significant displacement of the RS domain relative to the domain for RNA binding. So to gain an insight into the functional aspect of SRSF2 mutations, we performed sequencing analysis of mRNAs extracted from mutant (P95H) SRSF2-transduced HeLa cells in which expression of the wild-type and mutant SRSF2 were induced by doxycycline. The abnormal splicing in mutant SRSF2-transduced cells was directly demonstrated by evaluating the read counts in different fractions. Next, to investigate functional role of SRSF2 mutant, HeLa cells were transduced with lentivirus constructs expressing either the P95H SRSF2 mutant or wild-type SRSF2, and cell proliferation was examined. After the induction of gene expression, the mutant SRSF2-transduced cells showed reduced cell proliferation. In addition, we transduced P95H SRSF2 constructs into factor-dependent 32D cell lines. The expression of mutant SRSF2 protein resulted in increased apoptosis in the presence of IL-3 and also suppression of cell growth in the presence of G-CSF, which may be related to ineffective hematopoiesis, a common feature of MDS. To further clarify the biological effect of SRSF2 mutants in vivo, a highly purified hematopoietic stem cell population (CD34-c-Kit+ScaI+ Lin-) prepared from C57BL/6 (B6)-Ly5.1 mouse bone marrow was retrovirally transduced with either the mutant or wild-type SRSF2 with EGFP marking. The transduced cells were mixed with whole bone marrow cells from B6-Ly5.1/5.2 F1 mice, transplanted into lethally irradiated B6-Ly5.2 recipients, and we are now monitoring the ability of these transduced cells to reconstitute the hematopoietic system and other hematological phenotypes. Much remains, however, to be unrevealed about the functional link between the abnormal splicing of RNA species and the phenotype of myelodysplasia. Further functional studies should be warranted to understand these mechanisms in detail. In this meeting, we will present the results of our functional studies on the SRSF2 mutations and discuss the pathogenesis of MDS in terms of the alterations of splicing machinery. Disclosures: No relevant conflicts of interest to declare.

Identification and Characterization of a Novel Serine-Arginine-Rich Splicing Regulatory Protein

ABSTRACT We have identified an 86-kDa protein containing a single amino-terminal RNA recognition motif and two carboxy-terminal domains rich in serine-arginine (SR) dipeptides. Despite structural similarity to members of the SR protein family, p86 is clearly unique. It is not found in standard SR protein preparations, does not precipitate in the presence of high magnesium concentrations, is not recognized by antibodies specific for SR proteins, and cannot complement splicing-defective S100 extracts. However, we have found that p86 can inhibit the ability of purified SR proteins to activate splicing in S100 extracts and can even inhibit the in vitro and in vivo activation of specific splice sites by a subset of SR proteins, including ASF/SF2, SC35, and SRp55. In contrast, p86 activates splicing in the presence of SRp20. Thus, it appears that pairwise combination of p86 with specific SR proteins leads to altered splicing efficiency and differential splice site selection. In all cases, such regulation requires the presence of the two RS domains and a unique intervening EK-rich region, which appear to mediate direct protein-protein contact between these family members. Full-length p86, but not a mutant lacking the RS-EK-RS domains, was found to preferentially interact with itself, SRp20, ASF/SF2, SRp55, and, to a slightly lesser extent, SC35. Because of the primary sequence and unique properties of p86, we have named this protein SRrp86 for SR-related protein of 86 kDa.

SR-related proteins and the processing of messenger RNA precursors

The processing of messenger RNA precursors (pre-mRNA) to mRNA in metazoans requires a large number of proteins that contain domains rich in alternating arginine and serine residues (RS domains). These include members of the SR family of splicing factors and proteins that are structurally and functionally distinct from the SR family, collectively referred to below as SR-related proteins. Both groups of RS domain proteins function in constitutive and regulated pre-mRNA splicing. Recently, several SR-related proteins have been identified that are associated with the transcriptional machinery. Other SR-related proteins are associated with mRNA 3prime end formation and have been implicated in export. We review these findings and evidence that proteins containing RS domains may play a fundamental role in coordinating different steps in the synthesis and processing of pre-mRNA.Key words: SR protein, RNA polymerase, spliceosome, polyadenylation, nuclear matrix.