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Pathway of histone mRNA degradation: Oligouridylation followed by bidirectional decay

Dissertation
Author: Jr. Thomas Edward Mullen
Abstract:
Histone mRNAs are rapidly degraded at the end of S phase or when DNA replication is inhibited. Histone mRNAs end in a conserved stemloop rather than a poly(A) tail. Degradation of histone mRNAs requires the stemloop sequence, which binds the stemloop binding protein (SLBP), active translation of the histone mRNA, and the location of the stemloop close to the termination codon. In this thesis I present evidence that the initial step in histone mRNA degradation is the addition of uridines to the 3' end of the histone mRNA, both after inhibition of DNA replication and at the end of S-phase. Lsm1 is required for histone mRNA degradation and is present in a complex containing SLBP on the 3' end of histone mRNA after inhibition of DNA replication. I cloned degradation intermediates that had been partially degraded from both the 5' and the 3' end. RNA interference (RNAi) experiments demonstrate that both the exosome (3'-5') and 5' to 3' decay pathway components are functionally required for degradation. cRT-PCR experiments corroborate the findings from the functional RNAi experiments by providing direct evidence that individual histone mRNAs are degraded simultaneously 5' to 3' and 3' to 5' when DNA synthesis is inhibited. Finally, I present evidence that SLBP protein expression is required for proper regulation of histone mRNA degradation when DNA synthesis is inhibited, but that the underlying mechanism is due to nuclear retention of properly processed histone mRNA. The latter suggests that one of the critical functions of SLBP in human cells is the proper export of histone mRNA to the cytoplasm.

TABLE OF CONTENTS

LIST OF TABLES………………………………………………………………………….viii

LIST OF FIGURES…………………………………………………………………………ix

Chapter

I.

INTRODUCTION…………………………………………… ……………….1

The eukaryotic cell division cycle………………………………………….1

Histones are the architectural units of chromatin………………………..2

Chromatin assembly………………………………………………………..5

Replication - dependent histone mRNA metabolism……………………..6

Significance of maintaining proper histone protein levels……………..12

Histone mRNA degradation is a regulated process……………………14

Nonsense - mediated decay……………………………………………….19

General mechanisms of mRNA degradation……………………………21

Connection between histone metabolism and cell cycle chec kpoints.33

Summary……………………………………………………………………36

II.

HISTONE mRNA IS DEGRADED SIMULTANEOUSLY FROM BOTH THE 5’ AND 3’ ENDS……………………………………………..56

Introduction…………………………………………………………………56

Materials and Methods……………………………………………………59

Results………………………………………… ……………………………66

vii

Discussion………………………………………………………………….77

III.

DEGRADATION OF HISTONE mRNA REQUIRES OLIGOURIDYLATION…………………………………………………...105

Introduction………………………………………………………………..105

Materials and Methods…………………………………………………..108

Results………………………………………………………… ………….114

Discussion……………………………………………………………….. 121

IV.

EXPRESSION OF STEMLOOP BINDING PROTEIN (SLBP) IS CRITICAL FOR PROPER METABOLISM OF HISTONE mRNA, INCLUDING PROPER HISTONE mRNA TURNOVER……………...149

Introduction………………………………………………………………..149

Materials and Methods…………………………………………………..154

Results…………………………………………………………………….160

Discussion………………………………………………………………...171

V.

SUMMARY AND CONCLUSION……………………………………….191

Introduction………………………………………………………………..191

Lsm1 - 7: An unlikely player in histone mRNA degradation…… ……..192

Replication - dependent histone mRNA degradation: Evolutionary

remnants of bacterial mRNA degradation?……………………………194

Bidirectional decay in mammals: The predominant mechanism?.....195

A family of poly(A) polymerases / terminal uridyltransfera ses

exists in eukaryotes………………………………………………..........196

Summary.…………………………………………………………………204

REFERENCES…………………………………………………………………...213

viii

LIST OF TABLES

Table

1.

mRNA decay factors………………………………………………………46

2.

siRNA sequences…………………………………………… …………….83

3.

Oligonucleotides used in oligo(dA) RT - PCR detection of

oligouridylated histone H2A and H3 mRNA…………………………...127

4.

siRNA sequences used to knock down TUTases…………………….129

5.

Oligonucleotides for RT - PCR of endogenous TUTase mRNA……...131

6.

List of the Cid family of enzymes present in S. pombe ………………207

7.

List of poly(A) polymerase - associated domain (PAPD) - containing

proteins present in the human genome………………………………..209

ix

LIST OF FIGURES

Figure

1.

The 3’ end of repl ication - dependent histone mRNAs end in an

evolutionarily conserved stemloop……………………………………….38

2.

Cell cycle regulation of histone mRNA, SLBP mRNA and SLBP

protein……………………………………………………………………….40

3.

Model of histone mRNA metabolism…………………………………….42

4.

Model of histone mRNA degradation (2004)……………………………44

5.

Eukaryotic 5’ - 3’ mRNA degradation pathway…………………………..48

6.

Eukaryotic 3’ - 5’ mRNA degradation pathway by the exosome……….50

7.

Deadenylation - independent decapping and 5’ - 3’ mRNA

degra dation…………………………………………………………………52

8.

General mechanisms of endonucleolytic mRNA degradation………...54

9.

Knockdown of 3’hExo does not affect histone mRNA degradation…..85

10.

Effect of knocking down PTB and Lsm1………………………………...87

11.

Cell cycle ana lysis of Lsm1 and Upf1 knockdowns……………………89

12.

Effect of decapping complex and 5’ - 3’ exonuclease knockdown on

histone mRNA degradation……………………………………………….91

13.

Effect of knockdown of exosome components on histone mRNA

degradation………………………………………… ………………………93

14.

Summary of 5’ - 3’ decay and exosome experiments…………………...95

15.

Detection of the 5’ and 3’ ends of capped histone H3 mRNA

in vivo by cRT - PCR……………………………………………………..…97

16.

New histone H3 gene, HIST2H3D ……………………………………….99

17.

Detection of uncapped histone mRNA by cRT - PCR upon

inhibition of DNA synthesis reveals degradation intermediates

with nonencoded uridines……………………………………………….101

x

18.

Model of histone mRNA degradation…………………………………..103

19.

Detection of oligour idylated histone mRNAs after inhibition of

DNA synthesis………………………………………………………..…..133

20.

Oligo(dT) RT - PCR controls……………………………………………..135

21.

Detection of oligouridylated histone mRNAs at the end of S phase..137

22.

RNAi to 6 of 7 TUTases present in the human genome is effective

both at the mRNA and protein level…………………………………....139

23.

RNAi screen to seven TUTases present in the human genome

identifies two putative TUTases involved in histone mRNA

degradation……………………………………………………………….14 1

24.

TUTase - 1 and TUTase - 3 are predominantly localized to the

cytoplasm of HeLa cells…………………………………………………143

25.

Biochemical characterization of TUTase - 3……………………………145

26.

Current model of histone mRNA degradation…………………………147

27.

SLBP knockdow n cells are viable but grow at a slower rate than

control cells…………………………………………………………….....176

28.

RNAi to SLBP results in cell cycle defects and activation of Chk1

kinase……………………………………………………………………...178

29.

Histone mRNA levels in SLBP knockdown cells are reduced and

only a

small portion of the mRNA is misprocessed and

polyadenylated……………………………………………………………181

.

30.

Histone protein levels are decreased in SLBP knockdown cells

and the normal histone mRNA degradation response to HU is

impaired…………………………………………………………………...183

31.

Properly processed replication - dependent histone mRNAs are

localized to the endoplasmic reticulum………………………………..185

32.

Histone mRNA in SLBP knockdown cells is retained in the

nucleus……………………………………………… …………………….187

33.

Model of SLBP - dependent histone mRNA export…………………….189

xi

34.

Phylogenetic tree comparing the PAPD - containing proteins

present in the genomes of humans, budding yeast, and

fission yeast……………………………………………………………….211

xii

ABBREVIATIONS

mRNA

messenger RNA

SL

stemloop

SLBP

stemloop binding protein

RNAi

RNA interference

snRNA

small nuclear RNA

mRNP

messenger ribonucleoprotein

TUTase

terminal uridylyltransferase

PAP

poly(A) polymerase

HU

hydroxyurea

cRT - PCR

circularization RT - PCR

bp

basepairs

nts

nucleotides

CHAPTER 1

INTRODUCTION

THE EUKARYOTIC CELL DIVISION CYCLE

The eukaryotic cell faithfully replicates itself in a well characterized set of events termed the cell division cycle. A cell that is not dividing is termed to exist in a resting, G0 sta te. Once the proper stimuli prompt the cell to divide and duplicate itself, it enters the cell division cycle (or cell cycle). The cell cycle is separated into four major phases: the first growth phase (G1), the DNA synthesis phase (S), the second growt h phase (G2), and finally mitosis (M) where the newly replicated chromosomes are segregated into two daughter cells.

The cell cycle is a highly ordered process of events that ensures proper completion of each phase prior to beginning the subsequent phas e. Much of this regulation is accomplished by cyclin - dependent kinases (CDKs). Their regulated enzymatic activity functions to control cell signaling events that dictate the passage from each phase to the next. The dividing cell encounters a variety of endogenous (e.g. oxidative stress) and exogenous (e.g. ultraviolet and ionizing radiation) stresses that challenge the fidelity of the cell division cycle. The impact of the stress is most deleterious at the DNA level since this fundamental nucleic acid e ncodes the essential information of life and damage or alteration to it will affect, likely in the negative direction, the encoded information present within the subsequent

2

generations. Fortunately eukaryotes have evolved a complex molecular surveillance system known as DNA damage checkpoints (or cell cycle checkpoints) that exists to protect the integrity of DNA.

HISTONES ARE THE ARCHITECTURAL UNITS OF CHROMATIN

During each cell division a eukaryotic cell undertakes the arduous task of faithfully repl icating its DNA during S phase for later segregation into two daughter cells. Not only must the cell replicate the entire genome but it must also appropriately package the newly replicated DNA into chromatin. The cell accomplishes this by coordinating the metabolism of histone proteins with chromatin assembly and DNA synthesis.

Histone proteins play an essential architectural role in compacting DNA molecules into structures we have come to know as chromosomes. Nuclear DNA exists in various compacted st ates through the function of the various histone proteins that exist in the cell. The more extended form of DNA exists as nucleosomes where approximately 146 bp of DNA is wrapped twice around an octamer containining two copies of each of the four core his tone proteins H2A, H2B, H3 and H4. A linker histone protein, histone H1, associates with each nucleosome as well as a variable length of linker DNA. Nucleosomes can then be compacted into the higher order of condensation termed chromatin.

Two major cl asses of histone proteins exist. The first class encompasses the replication - dependent histone genes (H2A, H2B, H3, H4 and H1), which are encoded by over 75 genes and expressed strictly during the DNA replication phase

3

of the eukaryotic cell cycle (Marzluf f et al., 2002) . Two copies of each heterodimer consisting of both H2A:H2B and H3:H4 underlie the composition of the nucleosome. These heterodimers are deposited during the process of DNA replication in order to fill in the nucleosomal gaps that exist a s a consequence of the progressing replication fork. The replication - dependent histone genes are coordinately expressed and are clustered in close proximity to one another in the genome of metazoans (Wang et al., 19 96a; Wang et al., 1996b) . Two major clusters encompass these genes and are present on chromosomes 1 and 6 in humans. The major cluster on the short arm of chromosome 6 (HIST1) includes the majority of the H2A, H2B, H3 and H4 genes as well as all the H1 genes. There are actually two smaller clusters of replication - dependent histone genes on chromosome 1 (HIST2 and HIST3) that together encompass another several copies each of H2A, H2B, H3 and H4.

The second class of histone genes encodes the variant (o r replacement) histone genes where, in general, their expression is not restricted to S phase. These genes do not occur in clusters within the genome as do the replication - dependent histone genes. Examples of some histone variants include H2A.X, H2A.Z, H2ABbd, macroH2A, H3.3, and centromeric H3 (cenH3) (Malik and Henikoff, 2003) . The variant histone H2A.X varies from H2A at the C - terminus and is the target of post - translational phosphorylation in response to DNA damage (e.g. DNA double strand breaks, ioniz ing radiation) (Rogakou et al., 1998) . Studies on H2A.Z have revealed that this histone variant plays an important role in maintaining a transcriptionally active state of chromatin (euchromatin) (Li et al., 2007) . MacroH2A

4

is enriched in the chromatin of the X - inactivated chromosome in female mammals (Costanzi and Pehrson, 1998) . Histone variant H2ABbd (Barr body deficient) is depo sited within transcriptionally active chromatin and seems to be mutually exclusive to that of macroH2A (Gautier et al., 200 4) . Histone H3.3 is unusual in that it differs from the canonical H3 by only four amino acid positions. The precise function of these changes is still unclear but appear to affect the ability of H3.3 to be deposited into transcriptionally active sites o f chromatin (Malik and Henikoff, 2003) . Centromeric H3 (cenH3) is, as its name suggests, strictly localized to the centromeres of eukaryotic chromosomes and functions in packaging chromatin at these sites. Additionally, tissue specific histones exist such a s the testis specific histone H1t which is thought to play a role in meiotic events during spermatogenesis (Lin et al., 2004) . Finally, it is interesting to point out that histone variants exist only in H2A and H3, while both H2B and H4 are invariant. Evolutionary and structural determinants certainly underlie the existence of H2A and H3 variants but such a discussion is out of the scope of this introduction.

The S phase - restricted synthesis of the core histones is the main, gross difference distinguishing the expression of the replication - dependent histone genes

from the variant histone genes. The replication - dependent histone genes encode the only eukaryotic messenger RNAs (mRNAs) that are not polyadenylated. Instead, the mRNAs encoding the core histone genes end in a conserved stem - loop structure on their 3’ end (see below and Figure 1). The 3’ end unique to the replication - dependent histone mRNAs serves important roles in the post - transcriptional regulation of these genes, including processing and mRNA stability.

5

Variant histone genes are, like all other mR NAs, polyadenylated at the 3’ end and their post - transcriptional regulation is for all intents and purposes identical to other polyadenylated mRNAs.

CHROMATIN ASSEMBLY

Chromosome replication is a rapid, error - prone process involving the coordination of the DNA synthesis machinery (e.g. DNA polymerases, helicases, etc.) with the molecules involved in the various types of DNA repair. The DNA polymerases (DNA polymerase α /primase, δ , and ε ) at the replication fork also require that a delicate balance of histone proteins as well as chromatin assembly activity is present in order to ensure proper assembly of the newly replicated DNA into chromatin. During the process of DNA replication, a major chromatin assembly pathway exists that includes the heterotri meric chromatin assembly factor 1 (CAF - 1) complex. CAF - 1 is comprised of the p150, p60, and p48 subunits and together are present at replication foci within the nucleus of eukaryotic cells (Verreault et al., 1996) . CAF - 1 activity is restricted to DNA replication and CAF - 1 associates with proliferating cell nuclear antigen (PCNA), which acts as the processivity factor for the DNA polymerase at the replication fork (Groth et al., 2007) . It performs the initial step in chromatin assembly, deposition of the H3 - H4 tetra mer.

HIRA is a second chromatin assembly complex that also functions to deposit H3 - H4 tetramers, but it largely functions to assemble chromatin outside of S phase, or in a replication - independent manner. HIRA contains seven WD repeat motifs as well as a domain distinct to itself (HIRA domain) (Tagami et al., 2004) . The WD

6

repeats allow HIRA to interact with other proteins, particularly the histone variant H3.3. Chromatin assembly outside of S phase largely encompasses incorporation of histone variants as a consequence of local changes in chromatin due to transcription or normal histone protein turnover (Gunjan et al., 2005) . It is clear that HIRA prefers to interact with H 3.3 which is often associated with active chromatin domains. The reason for this specificity is not clear but recent evidence suggests that H3.3 - H4 histone dimers act as important intermediates in the replication - independent assembly of chromatin (Tagami et al., 2004) .

REPLICATION - DEPENDENT HISTONE mRNA METABOLISM

In this dissertation I examine specifically the molecular mechanisms contr olling the stability of replication - dependent histone mRNAs and therefore an overview of histone mRNA metabolism as well as general mechanisms of histone mRNA stability are in order. Additionally, general mechanisms of mRNA degradation will also be discus sed. The following sections examine these subjects in detail.

Transcription

The levels of histone mRNA are tightly coupled to DNA synthesis. When cells enter S - phase, the transcription of histone genes increase 3 - 5 fold (Figure 2) due to the phosphory lation of the positive transcription factor p220 NPAT (NPAT) by cyclin E/cdk2 (Ye et al., 2003b) . Cyclin E/cdk2 is an essential cyclin - dependent kinase that regulates multiple aspects of the transition from G1 to S phase of the cell cycle. These aspects include E2F1 - dependent transcription of genes involved in DNA

7

replication. NPAT is located within subnuclear structures termed Cajal bodies that a foundation of literature suggests is the primary site of histone gene transcription and pre - mRNA processing (Handwerger and Gall, 2006) . In support of its suggested role in histone gene transcription, NPAT and its subsequent phosphorylation at the G1/S transition, is required for proper accumulation of coilin (p80) foci, a major structural component of Cajal bodies (Ma et al., 2000) . Although the transcriptional contribution is significant to increasing histone mRNA levels, there are a number of post - transcriptional mechanisms that ultimately result in the 35 - fold increase in histone mRNA seen during S phase. These mechanisms include increased pre - mRNA processing and changes in mRNA half - life. As I will subsequently discuss, histone mRNA stability and translation are intimately connected.

Pre - mRNA Processing

There are two cis - acting elements in histone mRNA that contribute to the processing of the pre - mRNA. One is the 26 nucleotide (nt) stem - loop (SL) sequence that is present on the 3’ end of histone mRNA. The second is a purine - rich downstream sequence termed the histone downstream element (HDE) (Figure 1). The HDE is recognized through the complementary base pairing with the 5’ end of U7 snRNA, which is contained within the U7 snRNP. The U7 snRNP is composed of the U7 snRNA as well as a heteroheptameric set of Sm proteins (Marzluff and Duronio, 2002) . Typical splicesomal snRNPs cont ain SmD1, SmD2, SmB/B', SmD3, SmE, SmF and SmG. In contrast, the U7 snRNP contains Lsm10 and Lsm11 in place of SmD1 and SmD2 (Pillai et al., 2001) . The SL is recognized by a second trans acting factor, the stem - loop binding protein (SLBP). This protein is required for

8

histone pre - mRNA processing in Drosophila and human cel ls (Dominski et al., 1999; Sullivan et al., 2001) and additionally functions in histone mRNA metabolism including translation.

The two cis elements allow for an endonucleolytic cleavage of the pre - mRNA between the SL and HDE that results in the formation of the mature 3’ end of the message. Importantly, replication - dependent histone genes do not contain introns, therefore processing of histone pre - mRNA does not require the elaborate mechanisms of splicing. Instea d, the final step in processing of the message is the endonucleolytic cleavage which occurs at the end of a conserved ACCCA sequence following the SL. The endonuclease responsible for cleavage of the pre - mRNA has recently been identified through UV cross - linking studies as CPSF - 73 (Dominski et al., 2005a) . CPSF - 73 and the heat - labile factor Symplekin are core comp onents of the histone pre - mRNA processing apparatus (Kolev and Steitz, 2005) . Recent evidence from the Steitz lab suggests protein components of the splicesomal U2 snRNP plays a non - splicing role in stimulating U7 - dependent histone pre - mRNA processing (Friend et al., 2007) .

The SL, HDE, SLBP and U7 snRNP have an inter - dependent function in histone pre - mRNA processing. This relationship and the data supporting it are described in more detail in the Introduction of Chapter 5. A more relevant topic to this discussion, is how histone pre - mRNA processing contributes to the increase in histone mRNA levels when cells enter S phase. That is, if transcription contributes a 3 - 5 fold increase in histone mRNA levels, where does the addit ional 10 fold increase to give the final increase of 35 - fold originate?

9

Harris et al. conducted a convincing study where they set out to separate the contributions of transcription and pre - mRNA processing to the dramatic increase of histone mRNA levels at the beginning of S phase (Harris et al., 1991) . They constructed a transgene containing a constitutive U1 snRNA promoter, followed by the complete U1 coding region, and a normal histone 3’ sequence (U 5 H transgene). Using CHO cells synchronized by mitotic shake - off, they determined that the transgene was regulated normall y in a cell cycle regulated manner and responded to inhibition to DNA synthesis. More interestingly, they observed that the U 5 H transgene increased 10 - fold as cells entered S phase compared to the 35 - fold of a normal cloned H2A gene. Therefore, it was cl ear the 3.5 - fold difference was due to transcription while the 10 - fold effect was likely post - transcriptional. Indeed this was the case since using a different transgene construct that had two processing sites: a normal histone processing site and a down stream polyadenylation site. Therefore, the accumulation of each transcript reflected the efficiency of 3’ end formation. The larger transcript (polyadenylated) predominated in G1 while the smaller, histone 3’ end - containing transcript was restricted to S phase. The levels of the histone 3’ end transcript present between G1 and S phase was approximately an 8 - fold difference supporting the notion that as cells transit from G1 to S phase, histone pre - mRNA processing efficiency increases from 8 - 10 fold. T aken together, the processing efficiency of histone pre - mRNAs, like other pre - mRNAs, contributes a significant component in determining the abundance of the mature mRNA found in the cytoplasm. As we will soon discover below, in contrast to the contributio n of

10

transcription and processing, there is a remarkable decrease in the half - life of histone mRNA that is specific to the end of S and throughout G2 phase.

SLBP as the Post - Transcriptional Regulator of Histone mRNA

SLBP plays a major role in all aspe cts of histone mRNA metabolism. The level of SLBP mRNA does not significantly change throughout the cell cycle but at the beginning of S phase, translation is stimulated by an unknown mechanism resulting in a 20 - fold increase in SLBP protein (Figure 2) (Whitfield et al., 2000) . The increased levels of SLBP are thought to be the driving factor that stimulates histone pre - mRNA processing efficiency 8 - 10 fold. Following pre - mRNA processing, SLBP remains bound to the SL as the mature message is exported to the cytoplasm for translation (Figure 3).

SLBP does not contain a nuclear export signal but instead is exported while being bound to the RNA. The details of histone mRNA export have been investigated in both Xenopus oocyt es and Drosophila S2 cells and it is clear that the mRNA export receptor TAP is important for translocating the histone mRNP through the nuclear pore complex (Erkmann et al., 2005a) . Once exported, SLBP is required

for translation of the message (Sanc hez and Marzluff, 2002) . When histone mRNAs are degraded, SLBP is recycled back to the nucleus through interactions with the import receptors Importin α /Importin β

(Erkmann et al., 2005b) . SLBP contains a nuclear lo calization (import) signal and amino acid mutations within the C - terminus of SLBP abrogate binding to Imp α /Imp β .

The role of SLBP in histone mRNA translation has been studied in detail using the Xenopus oocyte system where transcription is inactive but t he translational

11

machinery is fully competent. Unlike humans, frogs have two forms of SLBP: xSLBP1 and xSLBP2. The latter is oocyte - specific and involved in translational repression of maternally stored histone mRNAs in frogs (Sanchez and Marzluff, 2002, 2004) . xSLBP1 functions analogously to human SLBP and its role in translation has been conclusively defined using an MS2 fusion protein tethering system. Recruitment of an MS2 - xSLBP1 to the 3’ end of an injecte d histone mRNA containing an MS2 stem - loop sequence (also on the 3’ end) is sufficient for robust stimulation of translation compared to a reporter control RNA (Gorgoni et al., 2005; Sanchez and Marzluff, 2002) . Re cently the lab has discovered a protein, termed SLIP1 (SLBP Interacting Protein 1), which interacts with both SLBP and the 5’ cap binding protein eIF4G. SLIP1 is involved in histone mRNA translation and likely bridges between SLBP and the 5’ end of the mR NA (Cakmakci et al., 2008) . In summary, SLBP plays an integral role in histone mRNA metabolism. Many of the functions of SLBP mirror that possessed by poly(A) binding protein (PABP), multiple molecules of which binds stoichiometrically to the approximately 200 nt polyadenylated 3’ end that is formed during the cleavage and polyadenylation of mRNAs. PABP is important in regulating translation initiation and is a major d eterminant of mRNA degradation.

Degradation of Histone mRNA

The final step in metabolism of an mRNA is degradation of the mRNA. Regulation of the half - life of an mRNA can be a critical regulatory step in gene expression. It provides the best mechanis m for irreversibly inactivating the translation of an mRNA, and hence halting synthesis of a particular protein until

12

more mRNA is synthesized. The half - life of the mRNA is a critical contributor to the steady - state level of an mRNA, and hence potentially as important as the rate of transcription of the gene in determining mRNA levels. The information for the half - life of an mRNA, and regulation of that half - life, is encoded in the mRNA, often in the 3’ untranslated region (3’ UTR), and is often mediated by proteins that interact with the 3’ UTR. Degradation of histone mRNA plays a critical role in regulation of histone protein biosynthesis, helping to maintain the balance between DNA and histone biosynthesis (Sariban et al., 1985) . Replication - dependent histone mRNAs are present in large amounts only in S - phase cells; they are rapidly synthesized just prior to entry into S - phase and rapidly degraded at the end of S - phase or when DNA synthesis is inhibite d. A more detailed discussion of the known mechanisms controlling histone mRNA stability and general mechanisms of mRNA decay are detailed in subsequent sections (see below).

Full document contains 249 pages
Abstract: Histone mRNAs are rapidly degraded at the end of S phase or when DNA replication is inhibited. Histone mRNAs end in a conserved stemloop rather than a poly(A) tail. Degradation of histone mRNAs requires the stemloop sequence, which binds the stemloop binding protein (SLBP), active translation of the histone mRNA, and the location of the stemloop close to the termination codon. In this thesis I present evidence that the initial step in histone mRNA degradation is the addition of uridines to the 3' end of the histone mRNA, both after inhibition of DNA replication and at the end of S-phase. Lsm1 is required for histone mRNA degradation and is present in a complex containing SLBP on the 3' end of histone mRNA after inhibition of DNA replication. I cloned degradation intermediates that had been partially degraded from both the 5' and the 3' end. RNA interference (RNAi) experiments demonstrate that both the exosome (3'-5') and 5' to 3' decay pathway components are functionally required for degradation. cRT-PCR experiments corroborate the findings from the functional RNAi experiments by providing direct evidence that individual histone mRNAs are degraded simultaneously 5' to 3' and 3' to 5' when DNA synthesis is inhibited. Finally, I present evidence that SLBP protein expression is required for proper regulation of histone mRNA degradation when DNA synthesis is inhibited, but that the underlying mechanism is due to nuclear retention of properly processed histone mRNA. The latter suggests that one of the critical functions of SLBP in human cells is the proper export of histone mRNA to the cytoplasm.