The Conserved Intronic Cleavage and Polyadenylation Site of CstF-77 Gene Imparts Control of 3′ End Processing Activity through Feedback Autoregulation and by U1 snRNP

<div><p>The human gene encoding the cleavage/polyadenylation (C/P) factor CstF-77 contains 21 exons. However, intron 3 (In3) accounts for nearly half of the gene region, and contains a C/P site (pA) with medium strength, leading to short mRNA isoforms with no apparent protein products. This intron contains a weak 5′ splice site (5′SS), opposite to the general trend for large introns in the human genome. Importantly, the intron size and strengths of 5′SS and pA are all highly conserved across vertebrates, and perturbation of these parameters drastically alters intronic C/P. We found that the usage of In3 pA is responsive to the expression level of CstF-77 as well as several other C/P factors, indicating it attenuates the expression of CstF-77 via a negative feedback mechanism. Significantly, intronic C/P of CstF-77 pre-mRNA correlates with global 3′UTR length across cells and tissues. In addition, inhibition of U1 snRNP also leads to regulation of the usage of In3 pA, suggesting that the C/P activity in the cell can be cross-regulated by splicing, leading to coordination between these two processes. Importantly, perturbation of CstF-77 expression leads to widespread alternative cleavage and polyadenylation (APA) and disturbance of cell proliferation and differentiation. Thus, the conserved intronic pA of the CstF-77 gene may function as a sensor for cellular C/P and splicing activities, controlling the homeostasis of CstF-77 and C/P activity and impacting cell proliferation and differentiation.</p></div

Gene Ontology terms enriched for upregulated and downregulated genes after CstF-77 knockdown.

<p>GO terms in the Biological Process category with <i>P</i><0.001 are shown. Redundant GO terms (more than 75% overlap in associated genes with GO terms having a more significant <i>P</i>) are not shown.</p

The intronic pA of CstF-77 gene is regulated by other C/P factors.

<p>(<b>A</b>) Effect of CstF-64 knockdown by siRNA on the CstF-77.S/CstF-77.L ratio. Top, Immunoblot (IB) showing knockdown efficiency; bottom, CstF-77.S/CstF-77.L ratio as measured by RT-qPCR. P-value (T-test) comparing knockdown and control siRNA samples is shown. (<b>B</b>) As in (A), except that knockdowns of CFI-25 (left), CFI-68 (middle), and CFI-59 (right) are shown. (<b>C</b>) As in (A), expect that knockdowns of CPSF-160 (left) and CPSF-73 (right) are shown. (<b>D</b>) Expression of isoforms P and S from various pRinG-77S-1690 reporter constructs in proliferating and differentiating (1 day of differentiation) cells. The PAS type and DSE type of each construct are indicated (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003613#pgen-1003613-g002" target="_blank">Figure 2C</a> for detail). The ratio of the log₂(P/S) value from proliferating cells to that from differentiating cells is indicated, where P represents the abundance of isoform P and S the abundance of isoform S. The difference in log₂(P/S) value (proliferating cells vs. differentiating cells) is statistically significant (<i>P</i><0.05, T-test) for all three constructs.</p

Gene Ontology terms enriched for genes with regulated 3′UTRs after CstF-77 knockdown.

<p>GO terms in the Biological Process category with <i>P</i><0.001 are shown. Redundant GO terms (more than 70% overlap in associated genes with another GO term having a more significant <i>P</i>) are not shown.</p

The intron 3 of human CstF-77 gene (<i>CTSF3</i>) has several unique, conserved features.

<p>(<b>A</b>) Gene structure of <i>CSTF3</i>, encoding the C/P factor CstF-77. Exons are numbered. A pA in intron 3 leads to APA isoforms 2 and 3 (isoform 3 has retention of intron 2). The conservation profile is based on vertebrate genomes. (<b>B</b>) Histograms of intron size, 5′SS maximum entropy (MaxEnt) score, and 3′SS MaxEnt score for all RefSeq-supported introns in the human genome. Red lines indicate introns 1–3 of <i>CSTF3</i>. For each feature of intron 3, its value and percent of introns with a lower value are indicated. (<b>C</b>) Schematic of intron 3 flanked by exons 3 and 4. (<b>D</b>) Density maps of introns in the human genome based on the 5′SS or 3′SS score of upstream exon and intron size. Intron density map is a heatmap showing distribution of introns with respect to two parameters (x-axis and y-axis). Left, 5′SS vs. intron size; right, 3′SS vs. intron size. Relative density is based on ratio of observed values to expected values (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003613#s4" target="_blank">Materials and Methods</a> for detail), and is represented by colors using the color scale shown on the top. (<b>E</b>) As in (D), except that downstream 5′SS and 3′SS scores were used. The intron 3 of <i>CSTF3</i> is indicated by a circled asterisk in each graph.</p

The intronic pA of <i>CSTF3</i> is involved in feedback autoregulation.

<p>(<b>A</b>) Effect of CstF-77 knockdown by siRNA on expression of CstF-77.L and CstF-77.S mRNAs. Top, time course of the protein level of CstF-77 after knockdown measured by Immunoblot (IB). Data were first normalized to GAPDH and then to the time 0 hr, as indicated below the image. Bottom, the effect of CstF-77 knockdown on the expression of CstF-77.L and CstF-77.S mRNAs. The time points are the same as those in (A). (<b>B</b>) Effect of CstF-77.S knockdown on expression of CstF-77.L. Left, knockdown of CstF-77.S as verified by qRT-PCR (both isoforms 2 and 3 are measured); right, effect of CstF-77.S knockdown on the expression of CstF-77.L. (<b>C</b>) Left, effect of CstF-77 overexpression on the expression of endogenous CstF-77.L and CstF-77.S mRNAs. Right, overexpression of exogenous CstF-77 as verified by IB using an antibody against the Omni tag of exogenous CstF-77. (<b>D</b>) Effect of CstF-77.L and CstF-77.S knockdowns on the P/S ratio of transcripts expressed from the reporter plasmid pRinG-77S-831. (<b>E</b>) Effect of CstF-77 overexpression on the P/S ratio of transcripts expressed from the reporter plasmid pRinG-77S-831. The expression levels of exogenous CstF-77 are the same as those in (C). (<b>F</b>) Time course for expression of endogenous CstF-77.L and CstF-77.S mRNAs and the CstF-77.S/CstF-77.L ratio after plating cells. All values were normalized to those at the 0 hr. All data on mRNA expression were based on RT-qPCR.</p

U1 snRNP regulates the usage of intronic pA of CstF-77 gene.

<p>(<b>A</b>) Regulation of expression of factors in U1 and U2 snRNPs in C2C12 differentiation. Expression analysis was based on a microarray dataset from the GEO database (GSE11415). Expression changes are represented by colors based on the scale shown at the bottom of the graph. (<b>B</b>) Effect of knockdown of U1-70K on the CstF-77.S/CstF-77.L ratio. Top, Immunoblot (IB) showing knockdown efficiency; bottom, CstF-77.S/CstF-77.L ratio as measured by RT-qPCR. P-value (T-test) comparing knockdown and control siRNA samples is shown. (<b>C</b>) and (<b>D</b>) As in (B), except that knockdowns of SF3B1 (C) and U2AF65 (D) are shown. (<b>E</b>) Inhibition of U1 snRNP interaction with 5′SS by U1D oligo. Sequence of the 5′ region of U1 snRNA is shown. The consensus sequence of the 5′SS of all RefSeq-supported human introns is represented by a sequence logo. U1D sequence is also shown (locked nucleic acid (LNA) residues are in uppercase and 2′-OMe RNA residues are in lowercase, see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003613#s4" target="_blank">Materials and Methods</a> for detail). (<b>F</b>) Effect of U1D on the CstF-77.S/CstF-77.L ratio. (<b>G</b>) Effect of U1D on the (isoform P)/(isoform S) ratio for pRinG-77S-1690. (<b>H</b>) The (isoform P)/(isoform S) ratios for pRinG-77S-1690 vectors with weak or medium strength 5′SS in proliferating and differentiating (1 day of differentiation) cells. See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003613#pgen-1003613-g002" target="_blank">Figure 2D</a> for 5′SS sequences. The ratio of the log₂(P/S) value from proliferating cells to that from differentiating cells is indicated, where P represents the abundance of isoform P and S the abundance of isoform S. The difference in log₂(P/S) value (proliferating cells vs. differentiating cells) is statistically significant (<i>P</i><0.05, T-test) for all two constructs.</p

Intronic C/P of human and mouse CstF-77 genes correlates with the global 3′UTR length.

<p>(<b>A</b>) Analysis of the CstF-77.S/CstF-77.L ratio and global 3′UTR length regulation (RUD) using microarray and RNA-seq data. cUTR, constitutive UTR; aUTR, alternative UTR. Correlation of the CstF-77.S/CstF-77.L ratio with global 3′UTR length was found in 17 human tissues and cell lines (<b>B</b>), 11 mouse tissues (<b>C</b>), and proliferating and differentiating C2C12 cells (<b>D</b>) as indicated by the R² values (linear regression). (<b>E</b>) Validation of CstF-77.S and CstF-77.L expression in proliferating and differentiating C2C12 cells by RT-qPCR (left). The normalized CstF-77.S/CstF-77.L ratio is also shown (right). P, proliferating; D1, differentiation for 1 day; D4, differentiation for 4 days. (<b>F</b>) Expression of CstF-77 protein in proliferating and differentiating C2C12 cells. The relative amount of CstF-77 is indicated. GAPDH was used as internal control.</p

Cell cycle genes are affected by CstF-77 knockdown.

<p>(<b>A</b>) Cumulative fraction for gene expression changes (siCstF-77 vs. siCtrl) of all genes analyzed (11,396 in total) or cell cycle genes (540 in total). (<b>B</b>) Validation of mRNA expression changes by RT-qPCR for several cell cycle-related genes after CstF-77 knockdown. P-values (T-test) are indicated. (<b>C</b>) mRNA expression of C2C12 differentiation marker genes in cells over-expressing CstF-77 after 2 days of differentiation. Values were normalized to that of vector only. P-values are based on the T-test.</p

Contribution of intron size, 5′SS strength, 3′SS strength, and pA strength to the usage of the intronic pA of <i>CSTF3</i>.

<p>(<b>A</b>) Schematic of the pRinG-77S vector containing the 5′ region of intron 3 with the pA and variable 3′ regions of intron 3. Two isoforms (isoforms S and P) can be generated as indicated. Northern blot probe and RT-qPCR primers used to examine the expression of isoforms S and P are indicated. pCMV, CMV promoter; RFP, red florescent protein; EGFP, enhanced green florescent protein; SV40 pA, SV40 polyA site; AAA_n, poly(A) tail. (<b>B</b>) Analysis of intron size. Left, Northern blot analysis of isoforms S and P expressed from the pRinG-77S vector containing different 3′ intron inserts as indicated in the graph; right, quantification of the Northern blot data. The R² of linear regression is indicated. (<b>C</b>) Analysis of pA strength. Left, mutations of pA sequence and Northern blot analysis of isoforms; right, quantification of the Northern blot data. The pA represented by diamond is the wild type. The R² values of linear regression lines are all >0.98. (<b>D</b>) Analysis of 5′SS strength. Top, wild type (WT) and mutant (MT) 5′SS sequences used in this study, with their strength analyzed by three methods, i.e., position specific scoring matrix (PSSM), ΔG for base pairing with U1 snRNA, and maximum entropy (MaxEnt). The percentile of the MaxEnt value based on all RefSeq-supported introns in the human genome is also shown. Bottom, Northern blot analysis of isoforms using different constructs. The constructs all have AAUAAA as PAS. (<b>E</b>) Analysis of 3′SS strength. Left, WT and MT 3′SS sequences and strength scores. The percentile value based on all RefSeq-supported introns in the human genome is also shown. Right, RT-qPCR analysis of isoforms S and P from the pRinG-77S vectors with WT or MT 3′SS. The R² values of linear regression lines are >0.99. HeLa cells were used for all the <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003613#pgen-1003613-g002" target="_blank">Figure 2</a> studies.</p