scholarly journals Phosphorylation of Threonine 61 by Cyclin A/Cdk1 Triggers Degradation of Stem-Loop Binding Protein at the End of S Phase

2008 ◽  
Vol 28 (14) ◽  
pp. 4469-4479 ◽  
Author(s):  
M. Murat Koseoglu ◽  
Lee M. Graves ◽  
William F. Marzluff
Keyword(s):  
Cell Cycle ◽  
Fusion Proteins ◽  
Regulatory Mechanism ◽  
Binding Protein ◽  
Cyclin A ◽  
S Phase ◽  
Mrna Levels ◽  
Histone Mrna ◽  
Stem Loop

ABSTRACT Histone mRNA levels are cell cycle regulated, and a major regulatory mechanism is restriction of stem-loop binding protein (SLBP) to S phase. Degradation of SLBP at the end of S phase results in cessation of histone mRNA biosynthesis, preventing accumulation of histone mRNA until SLBP is synthesized just before entry into the next S phase. Degradation of SLBP requires an SFTTP (58 to 62) and KRKL (95 to 98) sequence, which is a putative cyclin binding site. A fusion protein with the 58-amino-acid sequence of SLBP (amino acids 51 to 108) fused to glutathione S-transferase (GST) is sufficient to mimic SLBP degradation at late S phase. Using GST-SLBP fusion proteins as a substrate, we show that cyclin A/Cdk1 phosphorylates Thr61. Furthermore, knockdown of Cdk1 by RNA interference stabilizes SLBP at the end of S phase. Phosphorylation of Thr61 is necessary for subsequent phosphorylation of Thr60 by CK2 in vitro. Inhibitors of CK2 also prevent degradation of SLBP at the end of S phase. Thus, phosphorylation of Thr61 by cyclin A/Cdk1 primes phosphorylation of Thr60 by CK2 and is responsible for initiating SLBP degradation. We conclude that the increase in cyclin A/Cdk1 activity at the end of S phase triggers degradation of SLBP at S/G2.

scholarly journals Stem-Loop Binding Protein, the Protein That Binds the 3′ End of Histone mRNA, Is Cell Cycle Regulated by Both Translational and Posttranslational Mechanisms

2000 ◽  
Vol 20 (12) ◽  
pp. 4188-4198 ◽  
Author(s):  
Michael L. Whitfield ◽  
Lian-Xing Zheng ◽  
Amy Baldwin ◽  
Tomohiko Ohta ◽  
Myra M. Hurt ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Regulation ◽  
Binding Protein ◽  
S Phase ◽  
Mrna Processing ◽  
Mrna Levels ◽  
Histone Mrna ◽  
Stem Loop ◽  
Histone Mrnas ◽  
Cycle Regulation

ABSTRACT The expression of the replication-dependent histone mRNAs is tightly regulated during the cell cycle. As cells progress from G1 to S phase, histone mRNA levels increase 35-fold, and they decrease again during G2 phase. Replication-dependent histone mRNAs are the only metazoan mRNAs that lack polyadenylated tails, ending instead in a conserved stem-loop. Much of the cell cycle regulation is posttranscriptional and is mediated by the 3′ stem-loop. A 31-kDa stem-loop binding protein (SLBP) binds the 3′ end of histone mRNA. The SLBP is necessary for pre-mRNA processing and accompanies the histone mRNA to the cytoplasm, where it is a component of the histone messenger RNP. We used synchronous CHO cells selected by mitotic shakeoff and HeLa cells synchronized at the G1/S or the M/G1 boundary to study the regulation of SLBP during the cell cycle. In each system the amount of SLBP is regulated during the cell cycle, increasing 10- to 20-fold in the late G1 and then decreasing in the S/G2border. SLBP mRNA levels are constant during the cell cycle. SLBP is regulated at the level of translation as cells progress from G1 to S phase, and the protein is rapidly degraded as they progress into G2. Regulation of SLBP may account for the posttranscriptional component of the cell cycle regulation of histone mRNA.

scholarly journals Phosphorylation of Stem-Loop Binding Protein (SLBP) on Two Threonines Triggers Degradation of SLBP, the Sole Cell Cycle-Regulated Factor Required for Regulation of Histone mRNA Processing, at the End of S Phase

2003 ◽  
Vol 23 (5) ◽  
pp. 1590-1601 ◽  
Author(s):  
Lianxing Zheng ◽  
Zbigniew Dominski ◽  
Xiao-Cui Yang ◽  
Phillip Elms ◽  
Christy S. Raska ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Amino Acids ◽  
Posttranscriptional Regulation ◽  
Binding Protein ◽  
S Phase ◽  
Mrna Processing ◽  
Histone Mrna ◽  
Stem Loop ◽  
Histone Mrnas ◽  
Nuclear Extracts

ABSTRACT The replication-dependent histone mRNAs, the only eukaryotic mRNAs that do not have poly(A) tails, are present only in S-phase cells. Coordinate posttranscriptional regulation of histone mRNAs is mediated by the stem-loop at the 3′ end of histone mRNAs. The protein that binds the 3′ end of histone mRNA, stem-loop binding protein (SLBP), is required for histone pre-mRNA processing and is involved in multiple aspects of histone mRNA metabolism. SLBP is also regulated during the cell cycle, accumulating as cells enter S phase and being rapidly degraded as cells exit S phase. Mutation of any residues in a TTP sequence (amino acids 60 to 62) or mutation of a consensus cyclin binding site (amino acids 99 to 104) stabilizes SLBP in G2 and mitosis. These two threonines are phosphorylated in late S phase, as determined by mass spectrometry (MS) of purified SLBP from late S-phase cells, triggering SLBP degradation. Cells that express a stable SLBP still degrade histone mRNA at the end of S phase, demonstrating that degradation of SLBP is not required for histone mRNA degradation. Nuclear extracts from G1 and G2 cells are deficient in histone pre-mRNA processing, which is restored by addition of recombinant SLBP, indicating that SLBP is the only cell cycle-regulated factor required for histone pre-mRNA processing.

scholarly journals Drosophila Stem-Loop Binding Protein Intracellular Localization Is Mediated by Phosphorylation and Is Required for Cell Cycle-regulated Histone mRNA Expression

2004 ◽  
Vol 15 (3) ◽  
pp. 1112-1123 ◽  
Author(s):  
David J. Lanzotti ◽  
Jeremy M. Kupsco ◽  
Xiao-Cui Yang ◽  
Zbigniew Dominski ◽  
William F. Marzluff ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Rna Binding ◽  
Binding Protein ◽  
Intracellular Localization ◽  
Mrna Processing ◽  
Nuclear Targeting ◽  
Histone Mrna ◽  
Stem Loop

Stem-loop binding protein (SLBP) is an essential component of the histone pre-mRNA processing machinery. SLBP protein expression was examined during Drosophila development by using transgenes expressing hemagglutinin (HA) epitope-tagged proteins expressed from the endogenous Slbp promoter. Full-length HA-dSLBP complemented a Slbp null mutation, demonstrating that it was fully functional. dSLBP protein accumulates throughout the cell cycle, in contrast to the observed restriction of mammalian SLBP to S phase. dSLBP is located in both nucleus and cytoplasm in replicating cells, but it becomes predominantly nuclear during G2. dSLBP is present in mitotic cells and is down-regulated in G1 when cells exit the cell cycle. We determined whether mutation at previously identified phosphorylation sites, T120 and T230, affected the ability of the protein to restore viability and histone mRNA processing to dSLBP null mutants. The T120A SLBP restored viability and histone pre-mRNA processing. However, the T230A mutant, located in a conserved TPNK sequence in the RNA binding domain, did not restore viability and histone mRNA processing in vivo, although it had full activity in histone mRNA processing in vitro. The T230A protein is concentrated in the cytoplasm, suggesting that it is defective in nuclear targeting, and accounting for its failure to function in histone pre-mRNA processing in vivo.

In vitro evidence that LHRH stimulates the recruitment of prolactin mRNA-expressing cells during the postnatal period in the rat

1994 ◽  
Vol 12 (1) ◽  
pp. 107-118 ◽  
Author(s):  
A Van Bael ◽  
R Huygen ◽  
B Himpens ◽  
C Denef
Keyword(s):  
Cell Cycle ◽  
Cell Population ◽  
Blot Hybridization ◽  
Postnatal Period ◽  
S Phase ◽  
Female Rats ◽  
Mrna Levels ◽  
Dot Blot ◽  
Total Cell Population

ABSTRACT We have studied the effect of LHRH and neuropeptide Y (NPY) on prolactin (PRL) mRNA levels in pituitary reaggregate cell cultures from 14-day-old female rats, by means of in situ hybridization and Northern blot analysis. As estimated by computer-image analysis, addition of LHRH on day 5 in culture for 40 h resulted in a 37% increase in the total cytoplasmic areas of cells containing PRL mRNA, visualized using a digoxigenin-labelled PRL cRNA. The size of individual PRL-expressing cells was not influenced, nor was the content of PRL mRNA per cell. A similar effect of LHRH was found by dot blot hybridization of extracted RNA. PRL mRNA levels were not affected by NPY. LHRH induced a 29% increase in the number of PRL mRNA-expressing cells processing through the S phase of the cell cycle, visualized by the incorporation of [3H]thymidine ([3H]T) into DNA over 16 h. The fraction of [3H]T-labelled cells was 10–12% of the total cell population. NPY did not influence the number of [3H]T-positive cells expressing PRL mRNA, but completely blocked the effect of LHRH on the latter population. The present data suggest that LHRH, probably via a paracrine action of gonadotrophs, stimulates the recruitment of new lactotrophs, an action which is negatively modulated by NPY. Since the magnitude of this effect was the same in the total pituitary cell population as in cells processing through the S phase of the cell cycle and presumably mitosis, recruitment of lactotrophs seems to be based on differentiation of progenitor or immature cells into PRL-expressing cells, rather than on a mitogenic action on pre-existing lactotrophs alone.

scholarly journals Presence of a Poly(A) Binding Protein and Two Proteins with Cell Cycle-Dependent Phosphorylation in Crithidia fasciculata mRNA Cycling Sequence Binding Protein II

2004 ◽  
Vol 3 (5) ◽  
pp. 1185-1197 ◽  
Author(s):  
Bidyottam Mittra ◽  
Dan S. Ray
Keyword(s):  
Cell Cycle ◽  
Binding Protein ◽  
High Specificity ◽  
Binding Activity ◽  
Mrna Levels ◽  
Crithidia Fasciculata ◽  
Target Mrna ◽  
Cycle Dependent

ABSTRACT Crithidia fasciculata cycling sequence binding proteins (CSBP) have been shown to bind with high specificity to sequence elements present in several mRNAs that accumulate periodically during the cell cycle. The first described CSBP has subunits of 35.6 (CSBPA) and 42 kDa (CSBPB). A second distinct binding protein termed CSBP II has been purified from CSBPA null mutant cells, lacking both CSBPA and CSBPB proteins, and contains three major polypeptides with predicted molecular masses of 63, 44.5, and 33 kDa. Polypeptides of identical size were radiolabeled in UV cross-linking assays performed with purified CSBP II and 32P-labeled RNA probes containing six copies of the cycling sequence. The CSBP II binding activity was found to cycle in parallel with target mRNA levels during progression through the cell cycle. We have cloned genes encoding these three CSBP II proteins, termed RBP63, RBP45, and RBP33, and characterized their binding properties. The RBP63 protein is a member of the poly(A) binding protein family. Homologs of RBP45 and RBP33 proteins were found only among the kinetoplastids. Both RBP45 and RBP33 proteins and their homologs have a conserved carboxy-terminal half that contains a PSP1-like domain. All three CSBP II proteins show specificity for binding the wild-type cycling sequence in vitro. RBP45 and RBP33 are phosphoproteins, and RBP45 has been found to bind in vivo specifically to target mRNA containing cycling sequences. The levels of phosphorylation of both RBP45 and RBP33 were found to cycle during the cell cycle.

Use of a cell cycle mutant to delineate the critical period for the control of histone mRNA levels in the mammalian cell cycle

1984 ◽  
Vol 4 (11) ◽  
pp. 2364-2369
Author(s):  
A Artishevsky ◽  
A M Delegeane ◽  
A S Lee
Keyword(s):  
Cell Cycle ◽  
Critical Period ◽  
Fibroblast Cell ◽  
Temporal Analysis ◽  
S Phase ◽  
Temperature Sensitive ◽  
Mrna Levels ◽  
Histone Mrna ◽  
Mrna Accumulation ◽  
Mammalian Cell Cycle

Temporal analysis of DNA replication and histone mRNA accumulation in a hamster fibroblast cell cycle mutant (K12) showed that histone mRNA accumulates periodically during the cell cycle and reaches its highest level in the S phase. The direct correlation between the initiation of DNA synthesis and the accumulation of histone mRNA to high levels in S phase demonstrated the strict interdependence of these two events. Moreover, a critical period necessary for histone mRNA accumulation occurred late in G1 phase. If cells were incubated at the nonpermissive temperature during this critical period, the amount of histone mRNA remained at the basal level. Transcription rate measurements indicated that the triggering of histone mRNA synthesis occurred in late G1 and this mRNA was synthesized at its maximal rate 3 to 5 h before its peak of accumulation. However, if cells were prohibited from synthesizing DNA as a consequence of the temperature-sensitive block in G1, the synthesis of histone mRNA was not initiated.

Cell cycle regulation of mouse H3 histone mRNA metabolism

1984 ◽  
Vol 4 (1) ◽  
pp. 123-132
Author(s):  
R B Alterman ◽  
S Ganguly ◽  
D H Schulze ◽  
W F Marzluff ◽  
C L Schildkraut ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Steady State ◽  
Dna Synthesis ◽  
In Vitro Transcription ◽  
S Phase ◽  
Gene Probe ◽  
Histone Mrna ◽  
Mrna Content

The mechanisms responsible for the periodic accumulation and decay of histone mRNA in the mammalian cell cycle were investigated in mouse erythroleukemia cells, using a cloned mouse H3 histone gene probe that hybridizes with most or all H3 transcripts. Exponentially growing cells were fractionated into cell cycle-specific stages by centrifugal elutriation, a method for purifying cells at each stage of the cycle without the use of treatments that arrest growth. Measurements of H3 histone mRNA content throughout the cell cycle show that the mRNA accumulates gradually during S phase, achieving its highest value in mid-S phase when DNA synthesis is maximal. The mRNA content then decreases as cells approach G2. These results demonstrate that the periodic synthesis of histones during S phase is due to changes in the steady-state level of histone mRNA. They are consistent with the conventional view in which histone synthesis is regulated coordinately with DNA synthesis in the cell cycle. The periodic accumulation and decay of H3 histone mRNA appear to be controlled primarily by changes in the rate of appearance of newly synthesized mRNA in the cytoplasm, determined by pulse-labeling whole cells with [3H]uridine. Measurements of H3 mRNA turnover by pulse-chase experiments with cells in S and G2 did not provide evidence for changes in the cytoplasmic stability of the mRNA during the period of its decay in late S and G2. Furthermore, transcription measurements carried out by brief pulse-labeling in vivo and by in vitro transcription in isolated nuclei indicate that the rate of H3 gene transcription changes to a much smaller extent than the steady-state levels of the mRNA or the appearance of newly synthesized mRNA in the cytoplasm. The results suggest that post-transcriptional processes make an important contribution to the periodic accumulation and decay of histone mRNA and that these processes may operate within the nucleus.

scholarly journals Regulation of histone mRNA in the unperturbed cell cycle: evidence suggesting control at two posttranscriptional steps.

1991 ◽  
Vol 11 (5) ◽  
pp. 2416-2424 ◽  
Author(s):  
M E Harris ◽  
R Böhni ◽  
M H Schneiderman ◽  
L Ramamurthy ◽  
D Schümperli ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Half Life ◽  
S Phase ◽  
Protein Synthesis Inhibitor ◽  
Mrna Levels ◽  
Histone Mrna ◽  
Synthesis Inhibitor ◽  
Processing Efficiency ◽  
Histone 3 ◽  
Cycle Regulation

The levels of histone mRNA increase 35-fold as selectively detached mitotic CHO cells progress from mitosis through G1 and into S phase. Using an exogenous gene with a histone 3' end which is not sensitive to transcriptional or half-life regulation, we show that 3' processing is regulated as cells progress from G1 to S phase. The half-life of histone mRNA is similar in G1- and S-phase cells, as measured after inhibition of transcription by actinomycin D (dactinomycin) or indirectly after stabilization by the protein synthesis inhibitor cycloheximide. Taken together, these results suggest that the change in histone mRNA levels between G1- and S-phase cells must be due to an increase in the rate of biosynthesis, a combination of changes in transcription rate and processing efficiency. In G2 phase, there is a rapid 35-fold decrease in the histone mRNA concentration which our results suggest is due primarily to an altered stability of histone mRNA. These results are consistent with a model for cell cycle regulation of histone mRNA levels in which the effects on both RNA 3' processing and transcription, rather than alterations in mRNA stability, are the major mechanisms by which low histone mRNA levels are maintained during G1.

Regulation of histone mRNA in the unperturbed cell cycle: evidence suggesting control at two posttranscriptional steps

1991 ◽  
Vol 11 (5) ◽  
pp. 2416-2424
Author(s):  
M E Harris ◽  
R Böhni ◽  
M H Schneiderman ◽  
L Ramamurthy ◽  
D Schümperli ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Half Life ◽  
S Phase ◽  
Protein Synthesis Inhibitor ◽  
Mrna Levels ◽  
Histone Mrna ◽  
Synthesis Inhibitor ◽  
Processing Efficiency ◽  
Histone 3 ◽  
Cycle Regulation

The levels of histone mRNA increase 35-fold as selectively detached mitotic CHO cells progress from mitosis through G1 and into S phase. Using an exogenous gene with a histone 3' end which is not sensitive to transcriptional or half-life regulation, we show that 3' processing is regulated as cells progress from G1 to S phase. The half-life of histone mRNA is similar in G1- and S-phase cells, as measured after inhibition of transcription by actinomycin D (dactinomycin) or indirectly after stabilization by the protein synthesis inhibitor cycloheximide. Taken together, these results suggest that the change in histone mRNA levels between G1- and S-phase cells must be due to an increase in the rate of biosynthesis, a combination of changes in transcription rate and processing efficiency. In G2 phase, there is a rapid 35-fold decrease in the histone mRNA concentration which our results suggest is due primarily to an altered stability of histone mRNA. These results are consistent with a model for cell cycle regulation of histone mRNA levels in which the effects on both RNA 3' processing and transcription, rather than alterations in mRNA stability, are the major mechanisms by which low histone mRNA levels are maintained during G1.

scholarly journals HIRA, the Human Homologue of Yeast Hir1p and Hir2p, Is a Novel Cyclin-cdk2 Substrate Whose Expression Blocks S-Phase Progression

2001 ◽  
Vol 21 (5) ◽  
pp. 1854-1865 ◽  
Author(s):  
Caitlin Hall ◽  
David M. Nelson ◽  
Xiaofen Ye ◽  
Kayla Baker ◽  
James A. DeCaprio ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Ectopic Expression ◽  
Cyclin A ◽  
S Phase ◽  
Dependent Manner ◽  
Sequence Motifs ◽  
Histone Gene Expression ◽  
Phase Progression

ABSTRACT Substrates of cyclin-cdk2 kinases contain two distinct primary sequence motifs: a cyclin-binding RXL motif and one or more phosphoacceptor sites (consensus S/TPXK/R or S/TP). To identify novel cyclin-cdk2 substrates, we searched the database for proteins containing both of these motifs. One such protein is human HIRA, the homologue of two cell cycle-regulated repressors of histone gene expression in Saccharomyces cerevisiae, Hir1p and Hir2p. Here we demonstrate that human HIRA is an in vivo substrate of a cyclin-cdk2 kinase. First, HIRA bound to and was phosphorylated by cyclin A- and E-cdk2 in vitro in an RXL-dependent manner. Second, HIRA was phosphorylated in vivo on two consensus cyclin-cdk2 phosphoacceptor sites and at least one of these, threonine 555, was phosphorylated by cyclin A-cdk2 in vitro. Third, phosphorylation of HIRA in vivo was blocked by cyclin-cdk2 inhibitor p21cip1. Fourth, HIRA became phosphorylated on threonine 555 in S phase when cyclin-cdk2 kinases are active. Fifth, HIRA was localized preferentially to the nucleus, where active cyclin A- and E-cdk2 are located. Finally, ectopic expression of HIRA in cells caused arrest in S phase and this is consistent with the notion that it is a cyclin-cdk2 substrate that has a role in control of the cell cycle.

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