Thesis on "Biochemistry of HNRNA C. And HRALY in Cancer and Normal Cells Using Northern Blots Analysis"

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Chapter Four -- Results and Data Analysis

Chapter Five -- Discussion

Chapter Six -- Conclusions

Biochemistry of hnRNA C. And hRALY in Cancer and Normal Cells using Northern Blots Analysis

Chapter One: Introduction

Structure and Function of hnRNP Proteins

The structure and function of heterogeneous nuclear ribonucleoproteins (hnRNP) provide the basis for a growing body of research concerning ways to detect cancer early on so that existing treatments will be more effective and therefore improve clinical outcomes. There has been increasing interest in the scientific community concerning hnRNP proteins for this purpose; these are highly abundant proteins that bind to pre-mRNA in the nucleus. In this regard, Dreyfuss, Matunis, Pinol-Roma and Burd (1993) report that, "Messenger RNAs (mRNAs) are formed in the nuclei of eukaryotic cells extensive posttranscriptional processing of primary transcripts of protein-coding genes. These transcripts are produced by RNA polymerase II and are termed heterogeneous nuclear RNAs (hnRNAs), a historical term that describes their size heterogeneity and cellular location" (p. 290). To date, researchers have identified more than 20 different hnRNP proteins which have been designated using the letters A through U. based upon the extent of their migration on SDS-PAGE gels (Dreyfull et al., 1993). Likewise, Ford, Wright and Shay (2002) note that, "The heterogeneous nuclear ribonucleoproteins (hnRNPs) are a large family of nucleic acid binding proteins that are often found in, but not restricted to, the 40S-ribonucleoprotein particle" (p. 580). Therefore, the ribonucleoprotein complex provides the framework in which the pre-mRNA is processed into mature messenger RNA (Ford et al., 2002).

The importance of RNA in protein synthesis relates to its potential to provide improved methods that can be used for the early identification of several types of cancer by measuring RNA amount of hnRNP C. And hRALY in both cancer and normal cells. In this regard, the functions of hnRNP C. include:

1. Pre-mRNA Packaging

2. Pre-mRNA splicing

3. Pre-mRNA transport

4. Regulate mRNA stability

5. Component of Telomerase

6. Regulation of IRES mediated Translation

7. Cancer Biology and hnRNP Proteins

8. hnRNP A and B. Proteins and Lung Cancer

It has been suggested that the distribution of hnRNPs on pre-mRNA results from their different binding specificities. However, the high concentration of the individual hnRNPs in the nucleus disputes this observation and suggests that the proteins are organized randomly based upon non-specific binding. Though their distribution on pre-mRNA remains a mystery it is clear that many of the hnRNP proteins are associated with pre-mRNA biogenesis. However, multiple functions have been proposed for most the hnRNPs and many of these remains controversial in the scientific community. For example hnRNP C, has been proposed to be involved in RNA splicing, RNA polyadneylation, regulation of mRNA stability, internal ribosome entry site mediated translation, and also has been shown to be a component of the telomerase holoenzyme. Likewise the hnRNP A and B. proteins have been shown to regulate alternative splicing and to be associated with mRNA transport from the nucleus, as well as regulating RNA stability. Several of the hnRNP proteins have been found to be integral components of telomers. In general a plethora of functions have been proposed for each of the individual hnRNP, too many to address in this thesis. According to Torosyan, Dobi, Glasman, Mezhevaya, Naga, Huang, Paweletz, Leighton, Pollard and Srivastava (2010), though, "Representing the most abundant nuclear proteins that are implicated in the spliceosome packaging of excised intron sequences, hnRNPs typically bind to exonic/intronic splicing silencers, thereby repressing splicing" (p. 2460). These researchers are quick to point out, though, that, "hnRNPs can hinder communication between factors bound to different splice-sites, thereby having a positive role in RNA splicing. Most importantly, the concentration of splicing factors can alter the kinetic equilibrium in splicing, resulting in changes in splice-site selection" (Torosyan et al., p. 2460). Other researchers have also identified these mechanisms. For instance, according to Rajan, Dalgliesh, Bourgeois, Heiner, Emami, Clark, Bindereif, Stevenin, Robson, Leung and Elliott (2009), "Active pre-mRNA splicing occurs co-transcriptionally, and takes place throughout the nucleoplasm of eukaryotic cells. Splicing decisions are controlled by networks of nuclear RNA binding proteins and their target sequences, sometimes in response to signaling pathways" (p. 1).

Associations between these proteins have also been investigated. For example, Mili, Shu, Zhao and Pinol-Roma (2001) report that, "In the cytoplasm, A1 is associated with its nuclear import receptor (transportin), the cytoplasmic poly (A)-binding protein, and mRNA. In the nucleus, A1 is found in two distinct types of complexes that are differently associated with nuclear structures" (p. 7307). The first class of the cytoplasm A1 has the pre-mRNA and mRNA, and is the same as hnRNP complexes that have been described in the research in the past; however, the other class of the cytoplasm A1 functions as freely diffusible nuclear mRNPs (nmRNPs) during the late nuclear maturation stages and the potential exists that it is linked with nuclear mRNA export (Mili et al., 2001).

These nmRNPs are distinguishable from hnRNPs because although they contain shuttling hnRNP proteins, the mRNA export factor REF, as well as mRNA, they do not contain pre-mRNA or nonshuttling hnRNP proteins (Mili et al., 2001). Significantly, nmRNPs also contain proteins that are not found in hnRNP complexes as well, including the alternatively spliced isoforms D01 and D02 of the hnRNP D. proteins, the E0 isoform of the hnRNP E. proteins, and LRP130; the latter isoform has also been described previously in the research and is a protein whose function remains unknown but seems to possess a unique sort of RNA-binding domain (Mili et al., 2001). According to these researches, "The characteristics of these complexes indicate that they result from RNP remodeling associated with mRNA maturation and delineate specific changes in RNP protein composition during formation and transport of mRNA in vivo" (Mili et al., 2001, p. 7307).

Studies have also shown that hnRNP proteins are concentrated in the nucleus when growth conditions are normal growth at which point they seem to be excluded from the nucleolus (Mili et al., 2001). In addition, it is known that a subset of the hnRNP proteins, hnRNPs A1 and K, shuttle between the cytoplasm and the nucleus constantly while others such as hnRNP C1/C2 and hnRNP U. do not exhibit this shuttling process and remain in the nucleus (Mili et al., 2001). Mediation of the nuclear export of hnRNP A1 is achieved through the function of a specific amino acid sequence known as M9; this amino acid satisfies the criteria for an authentic nuclear export signal (NES); in addition, M9 operates as the hnRNP A1 nuclear location signal via mediating binding of its nuclear import receptor, transportin (Mili et al., 2001). It is also known that hnRNP A1 keeps the capability to bind mRNA, temporarily, in the cytoplasm and most likely as its passes through the nuclear pore complex (NPC) as well (Mili et al., 2001). By contrast, hnRNP A1, hnRNP C1 and hnRNP C2 remain in the nucleus and the process for retention is mediated via another specific amino acid sequence contained in C. proteins that operates as a nuclear retention sequence (NRS) (Mili et al., 2001). Significant as well is the fact that this NRS is capable of taking precedence over NESs; consequently, it is thought that the removal of hnRNP proteins that contain NRS from mRNA is required in order for the nuclear export of mRNA to proceed (Mili et al., 2001).

Not surprisingly, other researchers have also investigated the recent trends in this area. In this regard, Martinez-Contreras,… READ MORE