BIOL 243: Human CNOT3 Take-Home Assignment (2021) – 12.5% of course grade Page 1 of 3 Molecular Genetic Analysis of Human CNOT3, a Component of the mRNA Deadenylase Complex (Due on Wednesday February...

All questions.


BIOL 243: Human CNOT3 Take-Home Assignment (2021) – 12.5% of course grade Page 1 of 3 Molecular Genetic Analysis of Human CNOT3, a Component of the mRNA Deadenylase Complex (Due on Wednesday February 24th, 2021 before 11:59 PM) The purpose of this take-home assignment is to review and reinforce some of the concepts of molecular genetics learned in BIOL 243 and to develop critical thinking skills Background Information: The CNOT3 gene is highly conserved in all eukaryotes and encodes a component of the Ccr4- Not deadenylase that degrades specific cytoplasmic mRNAs by removing the poly-A tail. Removal of the poly-A tail at the 3’ end of mRNAs by CNOT3 causes their degradation by 3’-5’ exoribonucleases. Recently, mutations in the CNOT3 gene have been linked to autism spectrum disorder (ASD) [Meyer et al. (2020) Clinical Genetics 98: 408-412]. Individuals containing a non-functional CNOT3 allele exhibit ASD phenotypes including a substantial delay in speech development. It is hypothesized that CNOT3-linked ASD is caused by aberrant regulation of genes involved in neuronal development. There are two alleles of the human CNOT3 gene. One allele (referred as WT or wild type) produces normal levels of CNOT3 mRNA or protein in the cell. In contrast, the other allele (referred as “ds”) does not produce any CNOT3 mRNA or protein. Figure 1. Left panel. CNOT3 is a component of the Ccr4- Not deadenylase that removes the poly-A tail of cytoplasmic mRNAs and promotes their degradation [Zheng et al. (2016) Stem Cell Reports 7: 90]. Right panel. Cytoplasmic localization of CNOT3 in human HeLa cells by immunofluorescence with a CNOT3 antibody (green) (CUSABIO TECHNOLOGY LLC). Instructions: • Type your answers to the following questions. Your responses must be double-spaced with 12-point Times, Times New Roman or Cambria font. Although there is no page limit for this assignment, your responses should be concise and clearly answer each question. • Your assignment must be in PDF format and submitted to the D2L (digital) Dropbox prior to 11:59 PM on Wednesday February 24th, 2021. • Do not submit this question sheet with your assignment. • Contact Dr. Chua if you have any questions about this assignment. • No citations or external resources other than the lecture notes and textbook readings are required for this assignment. • The work that you submit must be your own original work in your own words. Submissions that are identical or very similar are not acceptable and all suspected cases of academic misconduct are reported to the Head of the Department. See the University Calendar for more information on academic misconduct. BIOL 243: Human CNOT3 Take-Home Assignment (2021) – 12.5% of course grade Page 2 of 3 Questions: Q1. CNOT3 is a regulator of gene expression. What level of gene expression is being regulated by the CNOT3 protein and briefly explain why. (2 marks) Q3. Answer the following questions based on the transcript map in the figure above. Q3A. How many exons are found in the CNOT3 transcript? (1 mark) Q3B. How many introns are found in the CNOT3 transcript? (1 mark) Q3C. Which direction does transcription occur (L to R or R to L)? (1 mark) Q3D. Which direction does translation occur (L to R or R to L)? (1 mark) Q3E. Where would be the promoter located in this figure (L or R)? (1 mark) Q3F. Which letter on the transcript map corresponds to the transcriptional start site? (1 mark) Q3G. Which letter on the transcript map corresponds to the transcriptional stop site? (1 mark) Q3H. Which letter on the transcript map corresponds to the start codon? (1 mark) Q3I. Which letter on the transcript map corresponds to the stop codon? (1 mark) Q3J. Explain why the pre-mRNA is so much larger in nucleotides than the mature mRNA (2 marks) Q3K. Explain why the total length of codons in nucleotides does not correspond to the mature mRNA length? (2 marks) Q3L. Provide a brief definition (two sentences maximum) of the open reading frame. (2 marks) Q3M. A base substitution mutation in region “d” results in a stop codon. What is the effect of this mutation on the protein expression of CNOT3? (2 marks) Q3N. A base substitution mutation occurs in region “e”. What is the most likely effect of this mutation on the protein expression of CNOT3? (2 marks) % o f i nd iv id ua ls w ith de la ye d sp ee ch WT/WT WT/ds ds/ds Genotypes Ph en ot yp es Q2. The bar graph on the left shows the phenotypes (% of individuals exhibiting a delay in speech) of the three CNOT3 genotypes (WT/WT, WT/ds and ds/ds). Given the previous information describing the two types of alleles of the CNOT3 gene, explain how these three genotypes produce the distinct phenotypes. Your answer should also indicate how the relative levels of CNOT3 protein expression in each genotype contribute to the corresponding phenotype. (6 marks) a b c ed f g h Coding DNA strand Pre-mRNA: 14,310 base pairs Mature mRNA: 4,138 base pairs Protein: 753 amino acids There is a very thin dark line at “b” Ensembl Genome Browser [Cunningham et al. (2019) NAR 47: D745] Transcript map of precursor mRNA (pre-mRNA) encoded by the human CNOT3 gene BIOL 243: Human CNOT3 Take-Home Assignment (2021) – 12.5% of course grade Page 3 of 3 Q4. The nucleotide sequence of the CNOT3 gene from CNOT3ds/ds individuals was determined. The results showed that all nucleotide variations in CNOT3ds/ds individuals compared to CNOT3WT/WT individuals were found at the 5’ end of the transcriptional unit that prevented the attachment of the 5’-CAP to the CNOT3 mature mRNA. Provide an explanation how the nucleotide variations in the CNOT3 gene contribute to the phenotype of CNOT3ds/ds individuals. Your answer should not exceed four sentences. (4 marks) Q5. Several individuals with abnormally early onset of speech development were examined for nucleotide variations in the CNOT3 gene. All of the nucleotide variations were found in the promoter region of the CNOT3 gene. Provide an explanation how the nucleotide variations in the CNOT3 gene contribute to the phenotype of these individuals. Your answer should not exceed four sentences. (4 marks) Q6. It is hypothesized that CNOT3-linked ASD is caused by aberrant regulation of a subset of genes involved in neuronal development. Further understanding of CNOT3-linked ASD requires the identification of these neurodevelopment genes regulated by CNOT3. You conduct an experiment that examines and compares the poly-A tail lengths and abundance of mRNAs expressed in neuronal cells from CNOT3ds/ds and CNOT3WT/WT individuals. Explain how the neuronal genes regulated by CNOT3 would be identified from this experiment. Your answer should not exceed four sentences. (4 marks)