Gene expression in the eukaryotic cells

Gene expression is the process through which the genetic code contained in the nucleus is read and used by the cell. The expression of genes begins with the production of a single stranded RNA copy of one strand of the gene’s double helix.

This copying process is called transcription and is carried out by a RNA polymerase.

There are five classes of RNA, which play different roles within the cell. Nuclear ribosomal RNA (rRNA precursor. 4% out of total cell RNA), cytoplasmic rRNA, which is packaged in the nucleolus with ribosomal proteins to constitute the ribosomes (71%), heterogeneous nuclear RNA (hnRNA) which is considered to be the precursor of messenger RNA (mRNA) [7%], cytoplasmic mRNA which contains the information for protein translation (3%) and transfer RNA (tRNA) which carries the amino acids for the protein synthesis [15%] (ALBERTS et al. 1994e). RNA transcription is carried out by three different RNA polymerases in eukaryotic cells, polymerase I, II and III.

RNA polymerase I makes the large rRNA. RNA polymerase II transcribes genes whose RNAs are translated into proteins. RNA polymerase III produces a variety of very small, stable RNAs including the small 5S ribosomal RNA and transfer RNA (ALBERTS et al. 1994e).

Transcription is initiated when a RNA polymerase binds to the DNA in the region called promoter located approximately 30 base pairs upstream from the transcription initiation site of the region which actually encodes the functional RNA product. This region is called the promoter sequence because it is the site where various control factors are able to bind to prevent or promote access by the RNA polymerase. It usually contains a sequence known as the TATA box which is rich in the nucleotides T and A (ALBERTS et al. 1994e; SOLOMON et al. 1996b).

Initiation of transcription is complex and mainly depends on the presence of transcription factors which must be assembled into a complex on the DNA at the promoter in order to recruit the RNA polymerase to this site. The factor which binds to the TATA box is known as the TATA binding protein TBP; it is only one subunit of the transcription factor complex. Transcription factor IID (TFIID) is the first to bind to the promoter. Once TFIID is bound, other transcription factors including TFIIB are

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incorporated to the complex. Additional factors include TFIIE, TFIIF, and TFIIH which act together to bring RNA polymerase II (Pol II) into a position where it can initiate transcription [Fig. 9] (AL KHOURI and PAULE 2002). After Pol II has been tethered to the promoter, it must be released from the complex to begin transcription. A key step in the initiation of transcription is carried out by TFIIH, one subunit of which is a protein kinase that phosphorylates Pol II. This step is thought to promote an open structure in the promoter region of the DNA which allows transcription to begin. The complexity of this multicomponent system affords rich possibilities for controlling the expression of each gene in a time and tissue specific manner (ALBERTS et al.

1994b).

Fig: 9. Assembly of the general transcription factors required for the initiation of transcription by RNA polymerase II.

In the first step, transcription factor IID (TFIID) binds specifically to a TATA sequence. Next, TFIIB joins the complex, followed by polymerase II escorted by TFIIF, TFIIE, and TFIIH which are incorporated into the complex.

In the presence of ATP, TFIIH phosphorylates Pol II, which promotes an open structure in the promoter region of the DNA and allows transcription to begin. Reproduced from ALBERTS et al. 1994b.

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RNA polymerase II opens a local region in the double helix to expose the nucleotides on a short stretch of DNA on each strand. One of the two exposed DNA strands acts as a template for complementary base-pairing with incoming ribonucleoside triphosphate monomers, two of which are joined together by the polymerase to begin a new mRNA chain. The RNA polymerase then moves downstream (towards the 3’

end of the mRNA or the 5’ end of the transcribed DNA strand) along the strand of the DNA forming a complementary sequence to that strand from RNA nucleotide monomers in the 5’ to 3’ direction. Eventually, the polymerase II crosses a signal region containing the cleavage signal AAUAAA. Although polymerase II continues to transcribe mRNA past this point, the mRNA transcript is cut at a point 10 to 30 nucleotides downstream from the cleavage signal (ALBERTS et al. 1994a). The sequence localized at the end of the mRNA molecule including the cleavage signal is called the untranslated region (UTR).

In eukaryotic cells the newly synthesized mRNA molecule (termed pre-mRNA) undergoes specific posttranscriptional modification and processing before it can be translated into protein. The first modification of the single stranded RNA begins when the growing RNA transcript is about 20 to 30 nucleotides long. At that point a protective cap is added on its 5’ end by addition of a methylated G nucleotide (7- methylguanosine). Eukaryotic ribosomes cannot bind to an uncapped message. A second modification is the result of the activity of poly(A) polymerase which adds 100 to 200 adenine nucleotides (the poly(A) tail) to the untranslated region at the 3’end of the mRNA molecule. This poly(A) tail, protects the mRNA against degradation [Fig.

10] (GANDOLFI and GANDOLFI 2001; SOLOMON et al. 1996c). The poly(A) tail is useful for laboratory purification of mRNA from the other types of RNA.

Complementary strands of poly(T) can be produced synthetically and attached to solid surfaces such as magnetic beads or synthetic fibers. The poly(T) is able to hybridize to the poly(A) tail of the mRNA and immobilize it (ALBERTS et al. 1994b).

When mRNA is initially produced, it includes segments called exons. These are the sequences which will eventually be translated into the protein sequence. Other segments called introns represent intervening sequences which may have transcription regulatory function but do not contain information for the formation of a

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protein. These intron sequences are present only in recently transcribed mRNA (pre-mRNA) within the nucleus and are spliced out of the pre-mRNA by cutting at sites at both ends of the intron and rejoining the remaining ends. The edited mature mRNA is then transported through the nuclear envelope into the cytoplasm where it is used by the ribosomes to produce proteins in the process termed translation [Fig 10]

(SOLOMON et al. 1996c).

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d) a)

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Fig: 10. Posttranscriptional modifications and processing of eukaryotic mRNA in the nucleus.

a) A DNA sequence containing both exons and introns is transcribed by RNA polymerase to make the primary mRNA transcript. As the pre-mRNA is synthesized the molecule is

“capped” in its 5’ end. b) The 3’ of the mRNA molecule is cleaved at a sequence that designates the poly(A) addition site. c) Introns are removed from the molecule and the exons are gluing together. d) The mature mRNA is transported through the nuclear envelope and into the cytoplasm to be used for protein synthesis. Reproduced from SOLOMON et al.

1996c.

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The spectrum of mRNA species in the nucleus and the cytoplasm is unique for each cell type and each point in development. It is estimated that a typical cell contains as many as 10,000 different RNA transcripts at any given time point. Regulation of RNA transcription is a fundamental process in differentiation. Regulation occurs at various points beginning with modification of the structure of the DNA helix to permit interaction of transcription factors with the promoter region to permit the binding of polymerase II. The next level of control is achieved by the process of splicing, in which the introns are removed. Transportation of edited mRNA from the nucleus to the ribosomes is a third control point. The rate of degradation of mRNA within the nucleus or cytoplasm is a fourth important mode of regulation. Control of degradation begins with protection by the poly(A) tail. When the tail is longer than 15 residues, the protein factor [poly(A) binding protein PABP] can bind the tail. PABP then binds to the cap at the 5’ end of the RNA and protects that end as well. One mechanism for degrading such RNA is inclusion of several repeats of the targeting sequence AUUUA which enhances loss of residues in the poly(A) tail which in turn causes loss of PABP protection. The cytoplasm contains several RNAses which can rapidly degrade unprotected RNA from both the 5’ and 3’ ends.

Finally gene expression can be controlled at translation initiation (ALBERTS et al.

1994b). Eukaryotic mRNAs have half-lives ranging from 30 minutes to as long 24 hrs;

the average half-life of an mRNA molecule in a mammalian cell is about 10 hrs (SOLOMON et al. 1996c). However, the mRNA half-life in oocytes is longer. In contrast to any other somatic cell, in the oocyte the interval between synthesis and use of RNA molecules can be several weeks. RNA molecules and proteins are accumulated in the ooplasm during the growth phase and are required to sustain the early stages of embryonic development prior to the onset of embryonic DNA transcription. A series of strategies including cytoplasmic regulation of the poly(A) tail length (mRNA poly and deadenylation), RNA localization and protein phosphorylation or dephosphorylation have been developed by the oocyte to store RNA and proteins in a quiescent form for subsequent use during oocyte maturation and early development (GANDOLFI and GANDOLFI 2001; CHIAN et al. 2003). Studies in oocytes from Xenopus laevis have demonstrated that mRNAs containing short

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poly(A) tails are translationally inactive. These mRNAs are activated by extension of their poly(A) tail at later stages of development (Fig. 11). Other group of mRNAs have normal length poly(A) tails which become enzymatically shortened and inactivated (RICHTER 1996; RICHTER 1999).

In bovine oocytes, polyadenylation of some important developmentally gene transcripts has been studied. For example, it has been shown that the length of the poly(A) tail of the genes encoding, connexin 43, glucose transporter type 1, heat shock protein 70, oct-4, plakophilin, and RNA poly(A) polymerase was shorter at the germinal vesicle stage than at metaphase II of in vitro matured oocytes (BREVINI-GANDOLFI et al. 1999). Recently, abnormal patterns of polyadenylation and deadenylation of specific mRNA maternal transcripts have been correlated with delayed cleavage and low developmental competence of bovine oocytes (BREVINI et al. 2002).

Fig: 11. Polyadenylation of c-mos mRNA during Xenopus laevis oocyte maturation.

Progesterone binds a putative cell surface receptor, which leads to a transient decrease in cAMP levels and the activation of Eg2 kinase, which phosphorylates the cytoplasmic polyadenylation element binding protein (CPEB) which forms a complex with polymerase A. Subsequently,

c-mos mRNA undergoes polyadenylation-induced translational activation. Newly synthesized Mos, serine/threonine kinase, activates MAP kinase kinase, which culminates in the activation of MPF. Active MPF phosphorylates a number of substrates, responsible of the oocyte maturation. Adapted from RICHTER 1999.

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RNA localization represents a further intriguing regulatory mechanism of meiotic maturation and early embryonic development (KING et al. 1999). It is related to the process of polarity observed in the oocytes of various lower species in which a morphogenetic gradient is created which determines the embryonic axis. This defines the unequal spatial distribution (anterior or posterior pole) of mRNA within the cytoplasm (ST JOHNSTON 1995). The localization of mRNA results in a subsequent spatial restriction in production of the encoded proteins within the oocyte, concentrating the protein at a specific intracellular site and preventing unwanted concentrations of the protein elsewhere (for review see LASKO 1999 and GANDOLFI and GANDOLFI 2001).

Oocyte maturation and development of preimplantation embryos have a profound need for synthesis of proteins for both housekeeping functions and cell specialization (HYTTEL et al. 2000b; SIRARD et al. 1989).

Through protein phosphorylation or dephosphorylation exerted by a large number of different protein kinases and protein phosphatases, the oocyte can activate or deactivate proteins and utilize them at specific time point during maturation and early development (CHIAN et al. 2003). The protein Mos studied in oocytes from Xenopus laevis is a good example of a molecule that can regulate post-translational control of oocyte development changes by phophorylation. Conversely, Mos itself is regulated by changes of phosphorylation (NISHIZAWA et al. 1992). It is a serine-threonine kinase necessary to initiate oocyte maturation. However, Mos is rather unstable and phosphorylation of specific serines contained in its structure is required for increasing its stability and preventing its degradation (GANDOLFI and GANDOLFI 2001;

NISHIZAWA et al. 1992). An example of dephosphorylation is found in the activation of MPF; in this case the phosphate group on the Cdc2 catalytic site is removed by Cdc25 phosphatase which activates the MPF to induce meiosis resumption in the oocyte (ALBERTS et al. 1994d).