After a eukaryotic gene is transcribed, the primary transcript is modified to protect it from degradation and target it for export into the cytoplasm and eventually translation to protein (see Fig. 1). These modifications generate the mature transcript and include capping, splicing, and polyadenylation. Capping occurs shortly after the start of transcription, when a modified guanine nucleotide is added to the 5′ end of the mRNA. This terminal 7-methylguanosine residue is necessary for proper attachment to the 40 S ribosome subunit during translation initiation. It also protects the RNA from endogenous ribonucleases that degrade uncapped RNA, which is often viral in origin. RNA polymerases do not terminate transcription in an orderly manner. They tend to be processive, yet the cell cannot tolerate a population of mRNAs that are enormous in size. Therefore mRNAs have a signal, the sequence AAUAA, that defines the end of the transcript. In general, ribonucleases cut mRNAs shortly after that signal, and a chain of several hundred adenosine residues, the poly(A), is added to the free 3′ transcript end. RNA cleavage and synthesis of the poly(A) tail require binding of specific proteins, including cleavage/ polyadenylation specificity factor (CPSF), cleavage stimulation factor (CstF), polyadenylate polymerase (PAP), polyadenylate binding protein 2 (PAB2), cleavage factor I (CFI), and CFII, that function to catalyze cleavage and protect the mRNA from exoribonucleases. The poly(A) tail increases RNA stability and assists in RNA export to the cytoplasm and translation. Mutations in the poly(A) signal can result in hematologic disease. For example, there are thrombophilic patients with a mutation in the polyadenylation signal in the prothrombin gene that increases the stabilization of this mRNA, resulting in higher prothrombin protein levels and increased thrombosis.

Fig1. OVERVIEW OF GENE EXPRESSION FROM DNA TO PROTEIN VIA RNA. Gene expression is a complex process requiring multiple and strictly regulated steps: transcription of the primary transcript, RNA maturation through capping, splicing and polyadenylation, export to the cytoplasm, and translation into protein.

Before the mRNA is exported from the nucleus to be translated into protein, introns must be removed and the exons reconnected (see Fig.1). In complex multicellular organisms such as vertebrates, introns are approximately 10-fold longer than the exons. The sequence and length of introns varied rapidly over evolutionary time. The process of removing introns, termed splicing, requires a series of reactions mediated by the spliceosome, a dynamic complex formed anew on its substrate from 5 snRNAs and approximately 100 proteins to form small nuclear ribonucleoproteins (snRNPs).[1] Recent critical advances in cryoelectron microscopy have shed light on the dynamics of assembly and disassembly of the spliceosome and the splicing process.[2] Canonical splicing uses the major spliceosome and accounts for more than 99% of splicing. The major spliceosome is composed of the nuclear active snRNPs U1, U2, U4, U5, and U6 along with multiple specific accessory proteins, such as U2AF and SF1. The spliceosome complex recognizes the dinucleotide GU at the 5′ end of an intron and an AG at the 3′ end (Fig. 2). Splicing can also occur cotranscriptionally and is regulated by chromatin factors that regulate transcription.[3] As transcription proceeds, an RNA lariat structure forms as intermediate, connecting intron ends, providing for both excision of the intron and proper alignment of the ends of the two bordering exons to allow precise ligation. When the intronic flanking sequences do not follow the GU-AG rule, noncanonical splicing removes these rarer introns with different splice site sequences using the minor spliceosome.[4] The same U5 snRNP is found in the minor spliceosome, in addition to the unique yet functionally simi lar U11, U12, U4atac, and U6atac. Furthermore, there are splicing mechanisms, including tRNA splicing and self-splicing, that function without use of the spliceosomal machinery. tRNA intron excision and exon ligation are catalyzed by protein-only complexes. tRNA introns typically interrupt the anticodon loop and must be removed so that the mature tRNA can properly function in protein translation.

Fig2. DESCRIPTION OF THE CANONICAL SPLICING JUNCTION, HIGHLIGHTING SPLICING FACTORS RECURRENTLY MUTATED IN HEMATOLOGIC MALIGNANCIES.

Splicing is central to the output of a diverse transcriptome and then proteome. Alternative splicing (AS) can enhance the versatility and diversity of a single gene. By alternatively excising different introns along with the intervening exons, a wide range of unique transcripts and proteins of differing sizes can be generated. These alternatives, termed isoforms, come from one gene that generates a variety of mRNAs with varying exon composition. AS is widespread in complex Recognition of splicing sites is mediated by small conserved motifs: a GU dinucleotide at the beginning of the intron and an AG dinucleotide at the end, preceded by a polypyrimidine tract and a branch point A. Exons also contain functional sequences that promote splicing. All these motifs are bound by splicing factors that mediate the splicing process. Splicing factors highlighted in red are associated with hematologic malignancies. 3′SS, 3′ splice site; 5′SS, 5′ splice site; AML, acute myeloid leukemia; CMML, chronic myelomonocytic leukemia; ESE, exonic splicing enhancer; MDSs, myelodysplastic syndromes; poly Y tract, poly-pyrimidine tract; snRNP, small nuclear ribonucleoprotein.

multicellular organisms: the average human gene has seven different isoforms, and the number of known isoforms is rapidly increasing thanks to technologies such as long-read next-generation sequencing. AS is common and essential for the proper function of almost all hematopoietic cells. For example, B cells can produce both immunoglobulin M (IgM) and IgD at the same developmental stage using AS. Erythrocytes use AS to produce differing isoforms of cytoskeletal proteins. However, AS does not always give beneficial results.

One of the best examples of inappropriate splicing leading to hematologic disease is β-thalassemia, where several mutations that occur in the GU-AG splicing signals result in aberrant β-globin mRNAs. Abnormal splicing can also lead to AML, MDSs, and other hematologic disorders. Translocated in liposarcoma (TLS) is a protein that recruits splicing complexes to mRNAs and is involved in the TLS-ETS transcription factor ERG fusion oncogene in t(16;21) in AML. This fusion of TLS with the transcription factor ERG alters the splicing profile of immature myeloid cells, blocking the expression of genes required for proper differentiation. Trans-splicing is a form of splicing that joins two exons that are not within the same mRNA transcript. Some trans-splicing events occur when the intron splice sites are not filled by spliceosomes. Trans-splicing can lead to mRNAs displaying exon repetitions or chimeric fusion RNAs, which can mimic the presence of a chromosomal translocation in normal cells. For example, specific chimeric fusion mRNAs seen in acute leukemias, such as MLL-AF4, BCR-ABL, TEL-AML1, AML1-ETO, PML-RAR, NPM-ALK, and ATIC-ALK, have been found in blood cells of healthy individuals with normal chromosome karyotype. Interestingly, these individuals generally do not develop leukemia. In addition, in patients with chronic myelogenous leukemia (CML) resistance to tyrosine kinase inhibitor therapy has been linked to AS of the BCR-ABL transcript.

Splicing mutations in genes can occur in cis within the RNA itself as is the case for the aforementioned β-thalassemia, or in trans such as mutations in splicing factors or members of the spliceosome. Recurring mutations in several factors of the spliceosome result in MDSs and other hematologic malignancies (see Fig. 2).[5] Mutations in the splicing factor 3b, subunit 1 (SF3B1) have been observed in 68% to 75% and 81% of refractory anemia with ring sideroblasts (RARS) and RARS with thrombocytosis (T) patients, respectively, and result in alternative choice of the branchpoint site within the intron. Mutations in the U2 small nuclear RNA auxiliary factor I (U2AF1) and the serine/arginine-rich splicing factor 2 (SRSF2) result in sequence-specific splicing aberrations. U2AF1, a subunit of the U2AF heterodimer that also contains the polypyrimidine tract binding subunit U2AF2, carries distinct point mutations in its two zinc-fingers that contact the AG dinucleotide in the 3′ splice site. Mutations of U2AF1 S34 or Q157 residues create de novo 3′ splice site contacts that alter RNA splicing and result in preferential exon inclusion or exclusion, depending on the −3 or +1 nucleotide sequence at the 3′ splice site, respectively.[6] Mutations in U2AF1 are associated with a number of myeloid malignancies and occur in 8.7% to 11.6% of de novo cases of MDS. U2AF1-mutant MDS/AML cells exhibit enhanced stress gran ule response, pointing to a novel role for biomolecular condensates in adaptive oncogenic strategies.[6] SRSF2 is a member of the serine/ arginine-rich pre-mRNA splicing factors. SRSF2 recognizes so-called splicing enhancer sequences (ESE) within exons (see Fig. 2), with a 5′-SSNG-3′ consensus motif where S = C/G and N = C/G/T/U. Mutations of P95 in the RNA-binding domain of SRSF2 alter RNA binding and splicing, reflected in higher affinity for 5′-CCNG-3′ than 5′-GGNG-3′ containing exons and resulting in preferential inclusion of alternative exons containing CCNG-rich motifs and exclusion of exons containing GGNG-rich motifs.[7,8] Mutations in the SRSF2 gene are associated with MDS and related diseases, particularly CMML, with SRSF2 mutations reported in up to 47% of patients. Recent studies have identified numerous critical targets aberrantly spliced, such as the member of the PRC2 complex EZH2, involved in epigenetic regulation.12 In addition, it has been found that splicing factor mutations result in enhanced formation of DNA:RNA hybrids, so called R-loops, that induce DNA damage that promises to be exploitable in the treatment of these diseases.[9,10]

References

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[1] Wahl MC, Will CL, Lührmann R. The spliceosome: design principles of a dynamic RNP machine. Cell. 2009;136(4):701–718.

[2] Wilkinson ME, Charenton C, Nagai K. RNA splicing by the spliceosome. Annu Rev Biochem. 2020;89:359–388.

[3] Naftelberg S, Schor IE, Ast G, Kornblihtt AR. Regulation of alternative splicing through coupling with transcription and chromatin structure. Annu Rev Biochem. 2015;84:165–198.

 [4]  Patel AA, Steitz JA. Splicing double: insights from the second spliceosome. Nat Rev Mol Cell Biol. 2003;4(12):960–970.

[5] Yoshida K, Sanada M, Shiraishi Y, et al. Frequent pathway mutations of splicing machinery in myelodysplasia. Nature. 2011;478(7367):64–69.

 [6]  Biancon G, Joshi P, Zimmer JT, et al. Precision analysis of mutant U2AF1 activity reveals deployment of stress granules in myeloid malignancies. Mol Cell. 2022;82(6):1107–1122.

 [7]  Liang Y, Tebaldi T, Rejeski K, et al. SRSF2 mutations drive oncogenesis by activating a global program of aberrant alternative splicing in hematopoietic cells. Leukemia. 2018;32:2659–2671.

[8]  Kim E, Ilagan JO, Liang Y, et al. SRSF2 mutations contribute to myelodysplasia by mutant-specific effects on exon recognition. Cancer Cell. 2015;27(5):617–630.

[9]  Chen L, Chen JY, Huang YJ, et al. The augmented R-loop is a unifying mechanism for myelodysplastic syndromes induced by high-risk splicing factor mutations. Mol Cell. 2018;69(3):412–25.

[10]  Nguyen HD, Leong WY, Li W, et al. Spliceosome mutations induce R loop-associated sensitivity to ATR inhibition in myelodysplastic syndromes. Cancer Res. 2018;78(18):5363–74.

مواضيع ذات صلة

RNA Heterogeneity

Nuclear Export of RNA

The Alpha Helix, Beta Sheet, and Beta Turn

Structures within Proteins

تصميم البوادئ primer design

كيف نعرف تسلسل الجين

Control of Gene Function and Biochemical Activity in Cells

آليات إصلاح الحمض النووي المتضرر DNA repair mechanisms

Hydrophobic Forces

Hydrogen Bonds and the Chelate Effect

Genes in the Cell Nucleus Control Protein Synthesis

RNA Proofreading