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Thesis

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Shaun Gu

on 27 June 2013

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Transcript of Thesis


An autosomal recessive, neuromuscular disorder characterized by alpha motor neuron degeneration in the anterior horn of the human vertebrae

Phenotype Determined based on age of onset and degree of severity with many patients experiencing:

(1) Symmetrical muscular atrophy and fragility and subsequent motor impairment and paralysis.

(2) Death from respiratory distress
Previous Research
Identified that the Survival Motor Neuron (SMN) gene is located within the genome of all deuterostome animals such as vertebrates and echinoderms (Dillon-White 2012)

Duplications of the SMN gene were also found in a variety of surveyed vertebrates.
To perform a phylogenetic survey of SMN and SPF30 in non-deuterostome/non-protostome species including: anthropods, nematods, platyhelminthes etc in addition to previously gathered deuterostome sequences to determine how these genes have been evolving in juxtaposition to one another.
Methods
At the Genetic Level
Objective
A tBLASTn search was performed utilizing the cDNA_all option and the human SMN peptide sequence
Human and Drosophila SMN and SPF30 peptide sequences were obtained from the bioinformatics database ENSEMBL
Only those cDNA transcripts with E-Values less than 1.0 e-05 were used in this bioinformatics analysis
How Exactly was this Genomic Analysis of the Survival Motor Neuron Gene Performed?
Transcripts for each species were compiled into a comprehensive FASTA file including only the species' name, transcript sequence and Identification Code
Results
TreeDyn / PhyML Phylogenetic Tree

Provided a phylogenetic representation of SMN in our group of organisms

Platyhelminthes: There was no SMN gene in these species

Ixodes: The only non-deuterostome species to have an SMN gene duplication

PAML Output

Non-synonymous : Synonymous sequences was a ratio >1
tBLASTn
Utilizes a known peptide sequence and matches it to a translated DNA database
PAML
Phyogenetic Analysis of Maximum Likelihood

This phylogenetic program was used to determine the dN/dS values (ω) for the SMN and SPF30
dN/dS (ω)
Provides a statistical ratio between the non-synonymous sequences to synonymous sequences.
dN/dS
Provides a statistical ratio between the non-synonymous sequences to synonymous sequences.
dN/dS
Provides a statistical ratio between the non-synonymous sequences to synonymous sequences.
dN/dS
Provides a statistical ratio between the non-synonymous sequences to synonymous sequences.
Non-synonymous: A nucleotide mutation which produces an alteration in amino acid sequence.
Synonymous: A mutation where one nucleotide basepair is exchanged for another; no alteration occurs in the amino acid coding sequence
dN/dS is therefore a statistical measurement analyzing the rate at which a specific gene is evolving within a given taxa.
PAML
Represents the numerical amount of organisms for which a transcript for the SMN gene was searched
for.
Protostome
Non-Protostome Non-Deuterostome
Deuterostome
Source: Burghes AHM & CE Beattie. 2009. Spinal muscular atrophy: why do low levels of survival motor
neuron protein make motor neurons sick? Nat Rev Neurosci 10: 597-609.
snRNP Function:
Forms the spliceosome --> A structure responsible for "snipping" introns from pre-mRNA in eukaryotes.

SMN has an underlying function in alpha motor neurons that results in SMA when there is a loss of this gene

Conclusion


•The Survival Motor Neuron gene has evolved to become a dynamic, multifunctional, moonlighting protein responsible for one primary function (snRNP assembly) and several secondary functions as the result of weak negative selective pressures and correspondingly positive selective pressures as evidenced by its heterogenous ω values.

Genes characterized by heterogeneous ω values give rise to genes whose sequences evolve variably.

The variance in function and ω value in gene sequence across the six observed taxanomic groups is representative of the SMN's multifunctionality, in addition to being lost in several taxanomic groups, i.e. Platyhelminthes.

•In juxtaposition, the SPF30 gene has remained as a relatively static gene, responsible for one innate, primary function as evidenced by its relatively homologous ω value of near or close to zero, thus suggesting its evolutionary history as the result of negative selection.

In contrast, SPF30 has not been lost in any of the observed taxanomic groups, except for Equus callabus, which may be due to incomplete sequence on behalf of the genome database.

Generally, moonlighting proteins exist in contrast to the previous notion of the "One protein, one function" hypothesis.
Measured the value of ω at every codon position for a given taxonomic grouping to provide a comprehensive view of evolutionary selection within SMN & SPF30's sequences
Acknowledgments
Dr. Aram Stump: Research Thesis Advisor
Thesis Committee: Dr. Jonna Coombs and Dr. Deborah Cooperstein
Marsha Dillion-White: Provision of Collected Vertebrate Gene Sequences
Horace McDonnell: Support and Funding through Summer Fellowship Program
Current
Table 1. Species with other than one Smn gene. For species with two Smn genes, dS between those genes is shown. For the species with three, the average ± standard deviation for all dS values is shown.
Table 2. Species with other than one Spf30 gene. For species with two Spf30 genes, dS between those genes is shown. For the species with three, the average ± standard deviation of all pairwise dS values between genes is shown.
Table 1 represents those species which contained other than 1 SMN gene. The remaining 84 species surveyed each contained only a single SMN gene.

•Of the 105 animal species surveyed from Ensembl, a majority of them contained only one SMN and one SPF30 gene.

•Ten species displayed no evidence of the SMN gene within the peptide database:

Two Insects, One Nematode, all Five Platyhelminth species, and members of the Radiata and Porifera surveyed (Table 1).

•Eleven species, ten of which were vertebrates, displayed multiple copies of the SMN gene, with correspondingly low genetic distances among them annotated by their corresponding dS value (Table 1).
Results
•Only one species displayed no evidence of the SPF30 gene within the peptide database

Equus callabus (Table 2)

•Four species displayed multiple copies of the SPF30 gene, with correspondingly low genetic distances among them annotated by their corresponding dS value (Table 2).

•Species with the following criteria were excluded: [1] Incomplete Gene Sequence or Annotation [2] Gene-encoded Peptides Aligned with Significant Gaps [potentially due to evolutionary loss by the gene)
Results
Results
Figures 3a-f represents the PAML dN/dS estimation identified as ω for each of the SMN gene codon positions.
•Corresponding to preceding likelihood ratio test results, the primate sequence set displayed many sites with a ω>1; these sites were mostly clustered at its C-terminal end, especially near the Proline-dense, Self-association, Sm binding and hnRNP Q binding regions. (Figure 3a).

ω estimates for the remaining sequences (Figures 3b-f) displayed generally lower values with many of them close or at a value of 1.

Additionally, Figures 3b-f displayed highly conserved regions (reflected by ω very close to zero) near the N-terminus (responsible for GEMIN2 and/or RNA binding) as well as the C-terminus (responsible for Sm binding).

Generally, Tudor domain sites displayed somewhat high estimates of ω, although they tended to be lower than in terminal regions.
By comparison, Spf30 estimates for ω display a contrasting pattern in comparison to SMN.
(Figure 4).

Generally, the overall estimations for ω were exceptionally low, with most of them very close to zero with a few minor positions approaching or exceeding one (Figure 4 a,b,c and d).

Fish were the only exception to this pattern, displaying a number of sites with ω that approached or even surpassed one. (Figure 4e).

A consistent pattern in these five sequence sets was that the C-terminal portion of the sequence was very highly conserved in all five cases.
Figures 4a-e represents the PAML dN/dS estimation identified as ω for each of the SPF30 gene codon positions.
It was hypothesized that SMN has evolved as a moonlighting protein due to regions of relatively weak structural conservation acting on its sequence--resulting in the evolution of novel variants (Positive Selection) subsequently allowing the protein to perform additional supplementary functions
Hypothesis
•SMA results from the loss of a functional copy of the SMN1 geneΩΩ

•The disease results from low levels of SMN rather than an absence of the protein
SMN2
Humans possess an additional copy of the SMN gene, SMN2.
A missense mutation defined by a C->T nucleotide transition results in:

(1) An alteration in exonic splicing signal skipping exon 7 in 90% of transcripts
(2) Produces Smn protein with truncated
C-terminus--results in degradation.
References
Burghes AHM & CE Beattie. 2009. Spinal muscular atrophy: why do low levels of survival motor neuron protein make motor neurons sick? Nat Rev Neurosci 10: 597-609.

Coovert, D. D., Le, T. T., Mcandrew, P. E., Strasswimmer, J., Crawford, T. O., Mendell, J. R., Coulson, S. E., et al. (1997). The survival motor neuron protein in spinal muscular atrophy, 6(8), 21–22.

D’Amico, A., Mercuri, E., Tiziano, F. D., & Bertini, E. (2011). Spinal muscular atrophy. Orphanet journal of rare diseases, 6(1), 71. doi:10.1186/1750-1172-6-71

Dillon-White, M. (2012). Evolution of the survival motor neuron gene in the vertebrates. (Accession Order No. [1517874]

Lefebvre, S., Bürglen, L., Frézal, J., Munnich, a, & Melki, J. (1998). The role of the SMN gene in proximal spinal muscular atrophy. Human Molecular Genetics, 7(10), 1531–1536.

Lorson, C. L., Hahnen, E., Androphy, E. J., & Wirth, B. (1999). A single nucleotide in the SMN gene regulates splicing and is responsible for spinal muscular atrophy. Proceedings of the National Academy of Sciences of the United States of America, 96(11), 6307–11.


Additional references can be found on pages : 38-43
Significance
Table 2: Species with other than one SPF30 Gene
Table 1: Species with other than one SMN Gene.
A Phylogenetic Mapping of the SMN Moonlighting Protein and its Paralog SPF30
Presented by Shaun Gu

(Coovert et al 1997; D'Amico et al. 2011; Lefebvre et al. 1998; Lorson et al. 1999)
Introduction:
Spinal Muscular Atrophy and the Survival of Motor Neuron

Spinal Muscular Atrophy (SMA)
Other mutations include:
(1) Exclusion of exons 5 or 6, or simultaneously 5 and 7
(2) Chimeric Mutations: Fuse the 5' terminus of Smn1 to the 3' terminus of Smn2
(3) Gene Conversion: Pathogenic SMN allele converts Smn1 into Smn2
Hypotheses:
(1) Sm proteins B/B’, D1, D2, D3, E, F and G are bound to pICln

(2) They are methylated by PRMT5 and PRMT7 and then released and bound to the SMN complex
(3) snRNA transcribed in the nucleus is exported into the cytoplasm through various complexes and bound to the Sm-SMN complex by binding to the Gemin5 protein
(4) The SMN complex ads the Sm proteins to the snRNA where it is then methylated initiating their binding to snurportin and importin that mediates their nuclear localization
(5) The SMN complex with adjoining Sm proteins then localized within Cajal Body structures in the nucleus where they mature into the spliceosome.
Smn: A multifunctional protein
(1) Sm Protein Binding: (4) Neuronal Function:
(a) Tudor Domain (a) Tudor Domain
(b) C-Terminus

(2) Gemin2 Protein Binding:
(a) N-Terminus

(3)Oligomerization
(a) N and C Termini

There is compelling evidence that SMN is a moonlighting protein with a primary ancestral function in cytoplasmic snRNP assembly that has evolved to gain additional secondary functions
TranslatorX Server
(1) MUSCLE: Aligns AA sequences
(2) GBlocks: Takes MUSCLE output and removes poorly aligned AA sites.

The remaining sites were then reverse-translated back into their original codons
For each Species:
PAML
Used to estimate
dS values for species with more than one Smn gene
PAML was then used to create branch models which would determine what type of evolutionary selection has been acting on genes following duplication events.

This was done with Model A and Model B:

(1) Model A: Constraints determine likelihood that ω0=ω1

(2) Model B: determine likelihood that following duplication the branch ω1 would differ from all other branches ω0

Results
Full transcript