Determining specific protein involvement in the Bact to B* transition of

the yeast spliceosome

Ethan Hotson
Hunter College Biochemistry, mentored by Dr. Nancy Greenbaum
4/13/2017

Proposal Summary:

The spliceosome is a critical component in the process of transcription. Specifically,

spliceosomal activity within the cell plays a critical role in pre-mRNA splicing by removing

non-coding introns and ligating code-carrying exons. This is done through the complex and

varied process of spliceosome assembly, ligation, and disassembly, in which various snRNPs

(small nuclear ribonucleoproteins) bond, function and disassemble to perform pre-mRNA

splicing. As mistakes in this splicing can lead to disease, it is critical to identify the processes of

spliceosome assembly and function, to understand and ensure their success. The objective as

such is to ensure the success of the splicing of introns and the ligation of exons of pre-mRNA.

To do this, proteins found in the spliceosomal catalytic core assembly will be isolated, and used

to identify where and how they interact to promote or cause pre-mRNA splicing.

Background:

The spliceosome is a critical component of the process of transcription, the process by

which the genetic information carried by deoxyribonucleic acid, or DNA, is copied into single

stranded messenger ribonucleic acid, or mRNA. This copy of the original genetic information is

sent to the ribosome, which assembles proteins based on the template provided by DNA, and is

converted into a usable form by transfer RNA, or tRNA.

 The role of the major spliceosome in this process is simple; the ligation of exons, pieces

or transcribed RNA that contain relevant genetic information, by the removal of introns, buffer

regions of RNA that separate the relevant message regions of transcribed RNA, the

aforementioned exons. The chemistry of this reaction is not understood, but advances in the

technology used to analyze the structure and function of biochemical molecules has allowed

further study of this unknown chemical process.1 This has led to a better understanding of the

chemistry behind this molecule, and perhaps an understanding of how mistakes of the major

spliceosome can lead to problems. For example, failure of proper spliceosome activity can lead

to a variety of genetic diseases, either through long term mutation or the genetic change of

single cells. This can lead to cancers and other genetic diseases.2

One example of relatively recent and widely acknowledged feature of the nature of the

major spliceosomal catalytic core and its assembly, is the catalytically active U5 and U6 small

nuclear RNA complex. This portion of the process, a part of the spliceosomal assembly phase

of steps A and B (shown in Figure 1), is connected to the introduction of certain proteins, such

as Prp2 (also shown in Figure 1). Because of its critical part in the splicing step in major

spliceosome function, the Bact to B* step of catalytic activation has been researched in greater

depth in recent years.3

 Figure 1: Spliceosomal Assembly and Splicing process4

The steps of the process of major spliceosome assembly, location, function and

disassembly are extremely complex; the introduction and departure of many complex

RNA-protein molecules and individual proteins necessitates a complete list of steps to the

process.5 This transition from inactive catalytically to catalytically active is known as Bact-->B*, B

being the step of the spliceosome process in which the transition from non catalytically active to

catalytically active occurs, and named for the complex B which is formed at the beginning of the

step. Additionally, the significance of biomolecules with similar function are mentioned, such as

the group II self-splicing intron, which is of note because it presumably is able to perform the

same chemical reaction to separate introns from exons and ligate important RNA that the

spliceosome does, while being an intron in itself.

 Further relevant research to the current direction of major spliceosome biochemistry in

the Bact to B* provides more specific information about the biomolecules involved in each step of

the process. The Spliceosome: Design Principles of a Dynamic RNP Machine describes the

chemical parts of certain critical biomolecules, one example being U2 snRNA.3 The most major

named parts of spliceosome assembly, such as U2 and other U snRNAs, are composed of both

RNA and protein, but the specifics of these components are as yet unknown. Thus, it is

important to understand what chemical parts allow for the structurally and chemically dynamic

nature of these biomolecules. Additionally, the ability of these molecules themselves to change

form is addressed, such as the massive remodeling that occurs in U4/U6.U5 tri-snRNP to allow

it to become catalytically active and ligate. The U4 departs, while the U5 and U6 remain,

becoming able to perform the chemical reaction necessary. The most important implication of

understanding this process is the prediction or understanding of mistakes made by the

spliceosome.

Finally, the catalytic center of the spliceosome is critical to an understanding of the

process by which the spliceosome forms and functions. The U2/U6 spliceosomal catalytic center

of the active spliceosome is the functional region which performs the splicing and ligation.6 For

this reason, a significant portion of current research is going toward the understanding of this

process, which is the reason the spliceosome assembles in the first place. It is also one of the

most complicated steps of the process, and requires the introduction of many proteins.

Project Description:

This project focused on understanding the role of a specific to be determined protein

within the process of spliceosome assembly. Specifically, there will be two goals to the project:

determining the mechanism by which the transition to catalytically active state (in the Bact to B*
transition) occurs and the dynamics of the resulting shift in catalytic core functionality by way of

certain proteins identified in the process. This portion, the catalytic core of U2-U6 snRNA is

naturally dormant, unable to splice even when assembled. Only when certain proteins, such as

the currently being studied Cwc 27 and p147 parts, are added to the system does the catalytic

center become active. As such, the project will focus on determining how this selected proteins

chemistry allows it to perform its function. The result will not necessarily be a complete

understanding of the molecules chemistry, but rather an understanding of the part important to

such that is so critical to the activation of the spliceosomal catalytic center. This protein will be

chosen on a combination of three factors: its accessibility within the process (i.e. the required

effort to isolate and test the sample), the importance of its role in said process, determined by its

location within the process and other details of its role, and its complexity, as more simple

protein components will require less time to create structural analysis through X-Ray

crystallography.

Research Strategy:

To understand the dynamics of the spliceosomal catalytic core activation of Bact-->B*,

RNA and/or protein components of yeast spliceosomes will be site-specifically marked with

fluorescent dyes or gold nanoparticles. Determining which proteins to use require nondenaturing

gel electrophoresis. Samples of a mixture of U2 snRNA and specific proteins identified in the

process or U6 snRNA in a similar mixture will be ran using gel electrophoresis. The mixture

concentration will be determined using a spectrophotometer, then ran in a vertical non

denaturing polyacrylamide gel. Figure 2 displays a polyacrylamide gel, which separates bonded

and nonbonded RNAs and proteins through an electric charge from electrodes on opposite ends

of the gel. These electrodes attract them differently due to their different charges, separating
them (nucleic acids have a negative charge and proteins have a significantly weaker negative

charge).

Figure 2: Polyacrylamide gel running proteins9

The gel will be stained with two dyes, one that darkens when bonded to RNA, and another

which darkens when bonded to proteins. Each sample will be loaded in a separate lane, with

more than one sample being typically loaded in the same gel. If the areas of each lane which

darken in each color overlap significantly, the protein will clearly have bonded to the specific

snRNA component of the Bact spliceosomal catalytic core. When this protein(s) has been

determined, the protein will be ran in non-denaturing gel electrophoresis, to determine the

peptide count of the protein. This protein will be tested in conjunction with other elements of the

Bact stage spliceosome (not yet chosen), which will be mixed in small samples with the selected

protein(s). These samples will be run in a final gel. The proteins which are found to have

bonded in greatest numbers, determined by the size and boldness of the line when

photographed with a spectrometer, will be run in small amounts in an X-ray crystallography

assay or biomolecule Nuclear Magnetic Resonance Spectroscopy test, to determine the state of

the bonded parts of the spliceosome, providing information on their structure after the B* step

has been completed.

 Bibliography:
1
Will, C. L., & Luhrmann, R. (2010). Spliceosome Structure and Function. Cold Spring Harbor
Perspectives in Biology, 3(7). doi:10.1101/cshperspect.a003707

2
Zhou, J., & Chng, W.-J. (2017). Aberrant RNA splicing and mutations in spliceosome complex
in acute myeloid leukemia. Stem Cell Investigation, 4, 6.
http://doi.org/10.21037/sci.2017.01.06

3
Wahl, M. C., Will, C. L., & Lhrmann, R. (2009). The Spliceosome: Design Principles of a
Dynamic RNP Machine. Cell, 136(4), 701-718. doi:10.1016/j.cell.2009.02.009

4
Spliceosome [Digital image]. (2003). Retrieved January 8, 2017, from 2017, from
http://www.mpibpc.mpg.de/luehrmann

5
Ruby, S. W., Chang, T. H., & Abelson, J. (1993). Four yeast spliceosomal proteins (PRP5,
PRP9, PRP11, and PRP21) interact to promote U2 snRNP binding to pre-mRNA. Genes
& Development, 7(10), 1909-1925. doi:10.1101/gad.7.10.1909

6
Schmitzov, J., & Pena, V. (2012). Emerging views about the molecular structure of the
spliceosomal catalytic center. RNA Biology, 9(11), 1311-1318. doi:10.4161/rna.22359

7
Schmitzov, J., Rasche, N., Dybkov, O., Kramer, K., Fabrizio, P., Urlaub, H., . . . Pena, V.
(2012). Crystal structure of Cwc2 reveals a novel architecture of a multipartite
RNA-binding protein. The EMBO Journal, 31(9), 2222-2234. doi:10.1038/emboj.2012.58

8
Perea, W., Schroeder, K. T., Bryant, A. N., & Greenbaum, N. L. (2016). Interaction between the
Spliceosomal Pre-mRNA Branch Site and U2 snRNP Protein p14. Biochemistry, 55(4),
629-632. doi:10.1021/acs.biochem.5b01036

9
APA MLA Chicago Protein Electrophoresis [Jpg]. (2017). Bio Rad.