CSTF2

Cleavage stimulation factor 64 kDa subunit is a protein that in humans is encoded by the CSTF2 gene,which is located on the X-chromosome in Homo sapiens, but has an autosomal paralog on chromosome 19 coding for the protein CstF64τ.^[5]

CSTF2
Available structures PDB Ortholog search: PDBe RCSB List of PDB id codes 1P1T, 2J8P
Identifiers
Aliases	CSTF2, CstF-64, cleavage stimulation factor subunit 2
External IDs	OMIM: 300907; MGI: 1343054; HomoloGene: 80171; GeneCards: CSTF2; OMA:CSTF2 - orthologs
Gene location (Human) Chr. X chromosome (human)[1] Band Xq22.1 Start 100,820,359 bp[1] End 100,841,520 bp[1]
Gene location (Mouse) Chr. X chromosome (mouse)[2] Band X|X E3 Start 132,959,936 bp[2] End 132,987,568 bp[2]
RNA expression pattern Bgee Human Mouse (ortholog) Top expressed in oocyte secondary oocyte oral cavity gonad palpebral conjunctiva mucosa of pharynx testicle ganglionic eminence buccal mucosa cell epithelium of nasopharynx Top expressed in ventromedial nucleus dorsomedial hypothalamic nucleus paraventricular nucleus of hypothalamus lateral hypothalamus lateral septal nucleus dorsal tegmental nucleus anterior amygdaloid area ventral tegmental area arcuate nucleus subiculum More reference expression data BioGPS More reference expression data
Gene ontology Molecular function nucleic acid binding protein binding mRNA binding RNA binding Cellular component mRNA cleavage and polyadenylation specificity factor complex cleavage body nucleoplasm nucleus nuclear body Biological process mRNA splicing, via spliceosome termination of RNA polymerase II transcription mRNA processing mRNA 3'-end processing pre-mRNA cleavage required for polyadenylation tRNA splicing, via endonucleolytic cleavage and ligation mRNA polyadenylation Sources:Amigo / QuickGO
Orthologs Species Human Mouse Entrez 1478 108062 Ensembl ENSG00000101811 ENSMUSG00000031256 UniProt P33240 Q8BIQ5 RefSeq (mRNA) NM_001306206 NM_001306209 NM_001325 NM_001290398 NM_001290399 NM_133196 NM_001359053 NM_001359054 RefSeq (protein) NP_001293135 NP_001293138 NP_001316 NP_001293135.1 NP_001277327 NP_001277328 NP_573459 NP_001345982 NP_001345983 Location (UCSC) Chr X: 100.82 – 100.84 Mb Chr X: 132.96 – 132.99 Mb PubMed search [3] [4]
Wikidata
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This gene encodes a nuclear protein with an RRM (RNA recognition motif) domain. The protein is a member of the cleavage stimulation factor (CSTF) complex that is involved in the 3' end cleavage and polyadenylation of pre-mRNAs. Specifically, this protein binds GU-rich elements within the 3'-untranslated region of mRNAs.^[6][7]ition motif) domain. The protein is a member of the cleavage stimulation factor (CSTF) complex that is involved in the 3' end cleavage and polyadenylation of pre-mRNAs. Specifically, this protein binds GU-rich elements within the 3'-untranslated region of mRNAs.

This gene encodes a nuclear protein with an RRM (RNA recognition motif) domain. The protein is a member of the cleavage stimulation factor (CSTF) complex that is involved in the 3' end cleavage and polyadenylation of pre-mRNAs. Specifically, this protein binds GU-rich elements within the 3'-untranslated region of mRNAs.^[7]

CSTF-64kDa structure and function

Cleavage stimulation factor 2(CSTF2 or CSTF-64) is the second subunit of 3 assembled subunits making up the protein cleavage stimulation factor(CSTF). CSTF2 is assembled with cleavage stimulation factor 1(55kDa) and cleavage stimulation factor 3(77kDa) to generate a heterotrimeric CSTF complex. Cleavage stimulation factor is an important element of mRNA maturation due to its involvement in cleavage and polyadenylation. It does this by assisting and regulating in the process when assembled with other protein complexes. Cleavage stimulation factor 2 subunit specifically identifies the location where cleavage and polyadenylation will occur.^[8] When assembled, cleavage stimulation factor 2 binds to pre-mRNA towards the 3' end of the sequence. Cleavage stimulation factor 2 will bind to G/U rich portions of the 3' end of a mammalian RNA sequence because of its ribonucleoprotein-type RNA binding domain. This G/U rich portion of the sequence will notably be downstream of the RNA cleavage site.^[9] In order for the RNA cleavage to occur, multiple variations of polyadenylation machinery must also be recruited both upstream and downstream of the cleavage site in order to set polyadenylation in motion. In tandem with the cleavage and polyadenylation specificity factor complex(CPSF-160, CPSF-100, CPSF-73, CPSF-30, FIP1, WDR33), which binds upstream of the cleavage site, the cleavage stimulation factor will also assist in determining the length of the mRNA's poly-A tail through its other subunits.

The RNA recognition motif(RRM) identified in cleavage stimulation factor 2 is composed of multiple Beta(β)-sheets. This RNA recognition motif is located at the N-terminus of the protein.^[10] The central domain of this protein binds to guanine and uracil bases while differentiating between cytosine and adenosine residues. This allows cleavage stimulation factor to identify the U/G rich segment of DNA that is downstream of the cleavage and polyadenylation site. Cleavage stimulation factor 2, alongside its RNA recognition motif, contains a hinge domain and a C-terminal domain. The hinge domain binds cleavage stimulation factor 2 to the cleavage specificity factor subunit, symplekin. This hinge domain orients the complex to both the cleavage specificity factor complex as a whole, as well as the subunit cleavage stimulation factor 3. The hinge domain is made up of ~40-50 amino acid residues and allows cleavage stimulation factor 2 to change shape when binding to the U/G rich portion of the DNA sequence. The C-terminal domain remains largely under researched. When the cleavage specificity factor complex is bound to cleavage stimulation factor 3 and it is associated with the rest of the cleavage stimulation factor complex(through the hinge domain of CSTF2), it will move all complexes into the nucleus.^[11]

All three cleavage stimulation factors must be assembled in a complex for cleavage and polyadenylation to take place. Compared to cleavage specificity factor complex, CSTF acts as the functioning regulatory unit for the process. Without cleavage stimulation factor 2 bound to the U/G rich RNA sequence, no cleavage or polyadenylation will take place despite the complex not directly cleaving the RNA. This is the case for both standard cleavage and polyadenylation and for alternative cleavage and polyadenylation.^[12][8] Additionally, the C-terminal domain of cleavage stimulation factor 3 is responsible for altering RNA recognition of the RNA recognition motif, though it does not physically touch the RNA recognition motif of cleavage stimulation factor 2.^[8]

Cleavage stimulation factor 2, and with speculation, cleavage stimulation factor 3, both play roles in the histone processing heat-labile factor(HLF) that also assist in managing pre-mRNA cleavage. However, cleavage stimulation factor 2 is regulated by the cell cycle and when it is depleted in cells, the S-phase is slowed down.^[13]  This occurs because the maturation of mRNA is directly impacted by the cleavage and polyadenylation of the pre-mRNA. If cleavage stimulation factor 2 is not present to assist the HLF, cleavage stimulation factor complex, and cleavage specificity factor complex in recognizing and binding to the pre-mRNA sequence, then the cleavage and polyadenylation process will inhibit the completion of transcription. The rate of histone RNA processing(cleavage and maturation of non-polyadenylated RNA) is also impacted by the amount of cleavage stimulation factor 2 present in the cell as well. This occurs during the transition between the G1 phase into the S phase.^[13]

Cleavage stimulation factor 2 is also vital to the expression of genes that it regulates. Without its presence, cleavage stimulation factors 1 and 3 cannot regulate the expression of genes on it own due to its disassociation from the pre-mRNA sequence. But in the presence of cleavage stimulation factor 2, the expression of a gene increases exponentially due to the binding of cleavage stimulation factor 3.^[8] The RNA recognition motif domain present in cleavage stimulation factor 2 works in two ways. First, it recognizes the U/G rich downstream sequence element that the complex itself will bind to. Its second function is to associate with cleavage stimulation factor 3 to localize the spread of the full cleavage stimulation factor complex between the nucleus and the cytoplasm.

The RNA recognition motif in cleavage stimulation factor 2, and its bacterial counterpart, Rna15, do not bind to a strict consensus sequence in the downstream sequence element after the cleavage site. Rather, the RNA recognition motif binds based on changes in resonance structures of the proteins present in binding frames associating with the U/G rich sequence.^[14]

Cleavage stimulation factor 2 is roughly 2000~ base pairs long whilst excluding the polyadenylated region when isolated using HeLa cell DNA.^[15]  The protein structure of both Cleavage stimulation factor 3 and cleavage stimulation factor 2 in humans share homologies with both yeast and Drosophila when compared. This suggests that the structure and role of the subunits assembling the cleavage stimulation factor complex are highly conserved when contributing to the polyadenylation process. Cleavage stimulation factor 3 subunit in homo sapiens is incredibly similar to a Drosophila modifier gene; also insinuating that the genes encoding this protein complex play key roles in gene regulation and vertebrae cell growth.^[6][16] Both the cleavage stimulation factor complex and the cleavage specificity factor complex are cis transcription factors that are present in homo sapiens as well as many other mammalian species.

CSTF-64τ substitution

Cleavage stimulation factor 2(CSTF-64) has its paralog CSTF-64τ that is capable of identifying a cleavage site for alternative polyadenylation.^[10][5] There is overlap between both the general CSTF-64 transcription factor and its alternative. In alternative polyadenylation, the utility of affected proteins are changed because of the alteration to the mRNA sequence. CSTF-64τ also consists of a N-terminal, a C-terminal, a RNA recognition motif, a hinge domain, and a P/G rich domain.^[17] Cleavage stimulation factor 2 and CSTF-64τ differ in their affinity for binding to the symplekin protein complex involved in polyadenylation. Symplekin protein complex is a scaffolding nuclear protein required for recruiting other polyadenylation machinery to the cleavage site.^[18] Cleavage stimulation factor 2 binds with more affinity to the symplekin complex than CSTF-64τ. This difference in binding affinity is due to size and presence of the P/G rich domain present in the CSTF-64τ variant that block the hinge domain from binding to the symplekin complex.^[10] Both protein variants bind to U/G rich sequence elements downstream of the cleavage site and play important roles in regulating the identification of cleavage and polyadenylation.^[17] But both variations will negatively regulate each other.^[10][11]

If cleavage stimulation factor 2 in its original form is present in the nucleus, then it will bind to the rest of the cleavage stimulation factor complex and the U/G rich region. Though in instances of cleavage stimulation factor 2 absence, if CSTF-64τ is present, then it will bind instead.^[8]  Both paralogs are redundant in their ability to bind both the rest of the cleavage stimulation factor complex, and to the RNA sequence itself. CSTF-64τ can also regulate alternative cleavage and polyadenylation in the same manner that its original can.  However, if both CSTF-64τ and cleavage stimulation factor 2 are depleted in the cell then the rate of cleavage and polyadenylation will decrease exponentially.^[17]

Tissue specific expression

Mammalian testes

Cleavage stimulation factor 2 is encoded by a gene located on the X-chromosome, and coincidentally in testes of mammals, the gene will not be expressed. This is caused by X-inactivation in meiotic formation. Male mammals will not express the original copy of this gene due to presence of only one X chromosome. For that reason, the autosomal counterpart, CSTF-64τ, is expressed in mammal testes instead.^[19] However, without CSTF-64τ and its original counterpart then there is not sufficient machinery to support male spermatogenesis in mice due to specific development absences during spermatogenesis process. Mice with the original copy of the CSTF2 gene functioning were able to generate the protein cleavage stimulation factor 2.^[19] CSTF-64τ and cleavage stimulation factor 2 control very little gene expression when present or absent. However, in the genes they do regulate that have to do with spermatogenesis in mice, their absence decreases the expression of those genes significantly.^[19]

Mammalian brains

Three variations of cleavage stimulation factor 2 are found in mammalian brains, CSTF-64, CSTF-64τ, and βCSTF-64.^[12] Minor levels of CSTF-64τ are also found in immune cells. βCSTF-64 is important to neuron function due to its ability to add an additional 49 amino acids to the proline/glycine domain of cleavage stimulation factor 2. This variant is present in all vertebrates in all regions of the brain and peripheral nerves.^[20]  The βCSTF-64 variant originates from from tissue specific exons being included in the gene CSTF2 coding for the original variant, which is notably not from its autosomal paralog.  Instead, βCSTF-64 is encoded off of the X-chromosome in the same fashion that the original is as well. There is slight difference in polyadenylation efficiency between βCSTF-64 and CSTF-64, namely that βCSTF-64 responds less to stronger polyadenylation signals than CSTF-64 does. This provides a regulator to the alternative polyadenylation mechanism in exclusively the nervous system of vertebrates.^[20]

Mammalian immune cells

The amount of cleavage stimulation factor 2 present in a cell determines the kind of immunoglobin protein expressed in the cell. The kind of immunoglobin produced(membrane bound or secreted form) can alternate based on the presence of cleavage stimulation factor 2, which can switch expression from membrane bound to secreted form.^[21]  The selection of either an upstream or downstream cleavage and polyadenylation site determines the form of the immunoglobin protein. An upstream selection codes for a secreted form production, and a downstream selection codes for a membrane bound protein production.^[21]

Effects on alternative polyadenylation

Alternative polyadenylation is caused by the presence of one or more Poly A signal sequence. The polyadenylation site will have a stronger effect on gene expression if its affinity for binding transcription factors like cleavage specificity factor complex and cleavage stimulation factor complex.^[22] Polyadenylation and cleavage sites are determined in part by both the cleavage specificity factor complex and cleavage stimulation factor 2, which targets proximal potential sites containing the U/G rich region capable of binding to its RNA recognition motif.^[22] If both cleavage stimulation factor 2 and its CSFT-64τ variant are depleted, then there is a stronger selection for distal polyadenylation sites.^[23][24]

The subsequent alternative polyadenylation then causes lengthening or shortening of mature mRNA sequences that alter sequence elements like 3' untranslated regions(UTR). The variation of alternative polyadenylation has been linked to markers in certain stages of human cancers and other diseases.^[22] Specifically in colon cancer tissues, cleavage specificity factor 3 and cleavage stimulation factor 2 are expressed at a higher frequency than usual. 4 other cleavage and polyadenylation factors were over expressed in cancerous tissue as well, which differed from the normal regulation of alternative polyadenylation factor gene expression. The exact relation of these post-transcriptional factors to oncogenesis is not yet characterized.^[25]

Interactions

CSTF2 has been shown to interact with CSTF3,^[26] SUB1,^[27] SYMPK,^[26] BARD1^[28][29] and BRCA1.^[28][29]