Passage
Alzheimer disease (AD) is a progressive condition affecting memory and cognition that can be pathologically characterized by the presence of neurofibrillary tangles (NFTs) . NFTs, aggregates of hyperphosphorylated tau proteins that have dissociated from microtubules, are believed to contribute to neuronal degradation and subsequent AD symptoms. In general, ribosomes initiate translation of tau mRNA by binding the 7-methylguanosine (m7Gpp) cap present at the end of the 5′ untranslated region (UTR) of the mRNA molecule. However, researchers believe that tau protein translation initiation can occur without recognition of the m7Gpp cap. This alternate mechanism, referred to as cap-independent translation, indicates that ribosomes are recruited by internal ribosome entry sites (IRES) generally located in the 5′ UTR of the mRNA sequence.To determine the initiation mechanism of tau mRNA translation, neuroblastoma cells of AD mice were transfected with two different DNA constructs (Figure 1) . Construct 1 contained the 5′ UTR of tau mRNA isoforms (v1 and v2) extracted from cells displaying NFTs. Construct 2 included the 5′ UTR of the β-globulin gene, which normally codes for a plasma protein. The luciferase reporter genes, Renillla and Photinus, were also included in the two constructs.
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Figure 1 DNA constructs containing β-globulin or tau gene used to transfect neuroblastoma cellsLuciferase gene expression from the constructs was monitored in the absence and presence of a synthetic m7Gpp cap analog (Figure 2) . Translation of Renillla luciferase mRNA depended on ribosomal binding to the m7Gpp cap of the mRNA, but translation of Photinus luciferase required ribosome recruitment by an IRES in the 5′ UTR of the adjacent gene.
Figure 2 Luciferase activity in neuroblastoma cells exposed to m7Gpp cap analogIn a second study, researchers used siRNA to target initiation factor eIF4E, a peptide translated in a cap-dependent manner that facilitates the binding of ribosomes to the m7Gpp cap. Expression of factor eIF4E, elongation factor eEF2K, and tau v2 protein was assessed in siRNA-treated cells (Figure 3) .
Figure 3 Western blot of eIF4E, eEF2K, and tau v2 extracted from non-AD cells treated with eIF4E siRNA
Adapted from Veo BL, Krushel LA. Translation initiation of the human tau mRNA through an internal ribosomal entry site. J Alzheimers Dis. 2009; 16(2) :271-5.
-Assume gene expression of tau v1 and v2 are synchronized in AD mice cells, and tau mRNA levels are analyzed. Of the following, which technique would researchers use to identify the tau mRNA isoform with the longer half-life in the cytoplasm of AD mice cells?

Question

Passage
Alzheimer disease (AD) is a progressive condition affecting memory and cognition that can be pathologically characterized by the presence of neurofibrillary tangles (NFTs) . NFTs, aggregates of hyperphosphorylated tau proteins that have dissociated from microtubules, are believed to contribute to neuronal degradation and subsequent AD symptoms. In general, ribosomes initiate translation of tau mRNA by binding the 7-methylguanosine (m7Gpp) cap present at the end of the 5′ untranslated region (UTR) of the mRNA molecule. However, researchers believe that tau protein translation initiation can occur without recognition of the m7Gpp cap. This alternate mechanism, referred to as cap-independent translation, indicates that ribosomes are recruited by internal ribosome entry sites (IRES) generally located in the 5′ UTR of the mRNA sequence.To determine the initiation mechanism of tau mRNA translation, neuroblastoma cells of AD mice were transfected with two different DNA constructs (Figure 1) . Construct 1 contained the 5′ UTR of tau mRNA isoforms (v1 and v2) extracted from cells displaying NFTs. Construct 2 included the 5′ UTR of the β-globulin gene, which normally codes for a plasma protein. The luciferase reporter genes, Renillla and Photinus, were also included in the two constructs.
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Figure 1 DNA constructs containing β-globulin or tau gene used to transfect neuroblastoma cellsLuciferase gene expression from the constructs was monitored in the absence and presence of a synthetic m7Gpp cap analog (Figure 2) . Translation of Renillla luciferase mRNA depended on ribosomal binding to the m7Gpp cap of the mRNA, but translation of Photinus luciferase required ribosome recruitment by an IRES in the 5′ UTR of the adjacent gene.

Figure 2 Luciferase activity in neuroblastoma cells exposed to m7Gpp cap analogIn a second study, researchers used siRNA to target initiation factor eIF4E, a peptide translated in a cap-dependent manner that facilitates the binding of ribosomes to the m7Gpp cap. Expression of factor eIF4E, elongation factor eEF2K, and tau v2 protein was assessed in siRNA-treated cells (Figure 3) .

Figure 3 Western blot of eIF4E, eEF2K, and tau v2 extracted from non-AD cells treated with eIF4E siRNA
Adapted from Veo BL, Krushel LA. Translation initiation of the human tau mRNA through an internal ribosomal entry site. J Alzheimers Dis. 2009; 16(2) :271-5.
-Assume gene expression of tau v1 and v2 are synchronized in AD mice cells, and tau mRNA levels are analyzed. Of the following, which technique would researchers use to identify the tau mRNA isoform with the longer half-life in the cytoplasm of AD mice cells?

Quizplus · Accepted Answer

11ecba2d_0f6d_9fe0_a3bf_a79e3df2dc58_MD0008_00 During reverse transcription polymerase chain reaction (RT-PCR), the enzyme reverse transcriptase converts mRNA into double-stranded cDNA, which is then amplified in cycles to yield thousands of copies. The cDNA is initially denatured into single strands using heat, and forward primers and reverse primers anneal to the denatured cDNA strands so that Taq polymerase can elongate the DNA sequence. To identify which tau isoform has the longer half-life (ie, lasts longer in the cytoplasm) in the given scenario, the researchers must first convert tau v1 mRNA and tau v2 mRNA into cDNA. The number of amplified copies (concentration) of tau v1 cDNA can be compared with that of tau v2 cDNA by performing RT-PCR at varying time points. The tau isoform with greater cDNA concentration at the final time point has the longer half-life. By definition, each tau isoform mRNA differs in coding sequence; as a result, their cDNA sequences also differ. This outcome means that tau v1 and tau v2 cDNA molecules need different forward and reverse primers for the primer-annealing step of RT-PCR. (Choice A) Performing RT-PCR at only one time point would yield only one measurement, and would not allow continuous tracking of tau isoform mRNA levels in the cytoplasm. (Choices B and D) Western blot is used to detect proteins, not mRNA. Because the starting material is tau v1 and v2 mRNA, RT-PCR must be performed. However, a different technique that also could be employed in place of RT-PCR is northern blot, which is used to analyze mRNA expression levels. Educational objective: Researchers can assess the half-lifes of mRNA isoforms by converting mRNA to cDNA via RT-PCR at varying time points, allowing for comparison of isoform concentrations. The isoform with greater cDNA concentration at the final time point has the longer half-life.

Passage Alzheimer Disease (AD) Is a Progressive Condition Affecting Memory and and Cognition