RNA Interference and Recombination- Exam 3

steady state level of mRNA

amount of mRNA in cell available for translation

determined by combination of transcription and mRNA degradation rates

half-life of mRNA

mRNA is degraded at some point after synthesis

lifetime of mRNA varies; regulated by cell need

pathways of degradation

1. exoribonuclease enzymes shorten length of poly-A tail called deadenylation-dependent decay: binding of poly-A-binding protein to tail stabilizes mRNA

2. decapping enzymes removes 7-methylguanine cap: mRNA is now unstable

3. endonuclease cleaves mRNA internally

regulation of mRNA stability and degradation

adenosine-uridine rich element (ARE)

cis-acting sequence element that regulates mRNA stability

located in the 3’ untranslated region of mRNA molecules

RNA binding proteins (RBPs) bind to AREs and increase or prevent degradation

RNA interference (RNAi)

sequence specific post transcriptional regulation

short RNA molecules regulate gene expression in cytoplasm of plants, animals, and fungi; repress translation and trigger mRNA degradation

phenomena known as RNA-induced gene silencing

molecular mechanism of RNA-induced gene silencing

done by siRNA and microRNAs: short double-stranded ribonucleotides

siRNAs: arise in cell due to virus infection- produce double stranded RNA, which is recognized and cleaved by the enzyme dicer

microRNAs: noncoding RNAs that negatively regulate gene expression

siRNA pathway

double-stranded RNA is processed into siRNA by Dicer

siRNAs then associate with RISC containing an Argonaute (AGO) family protein

RISC unwinds the siRNAs into single-stranded siRNAs and cleaves mRNAs complementary to the siRNA

miRNA pathway

miRNA genes are transcribed as primary-miRNAs which are trimmed at the 5’ and 3’ ends by the nuclear enzyme Drosha to form pre-miRNAs

pre-miRNAs exported to the cytoplasm and processed by Dicer

miRNAs then associate with RISC and mRNAs

if the miRNA and mRNA are perfectly complementary, the mRNA is destroyed; if there is a partial match, translation is inhibited

non-coding RNAs

microRNAs- small noncoding fragments of RNA that regulate gene expression (thought to regulate at least 1/3 of all protein coding genes)

first discovered in C.elegans

attempts to inject long double stranded RNA molecules into worm cells was shown to disrupt translation in a sequence specific manner

is this a defense mechanism against RNA viruses

processing of siRNA and miRNA by Argonaut protein

both siRNA and miRNA are first trimmed by the Dicer protein in the cytoplasm

the smaller RNA fragments then bind to Argonaut

argonaut selects one of the two strands and then separates the unwanted strand which is then degraded

the other strand (guide strand) remains attached to Argonaut

additional proteins bind to form the RNA induced silencing complex (RISC)

guide strand recognizes a target sequence and the RISC then binds to it

RNA induced silencing complex (RISC)

double stranded micro RNAs and siRNAs are processed by enzyme dicer

dicer cleaves the RNA transcript into smaller fragments

effector complex proteins bind the cleaved double stranded RNA and make it single stranded

single strands form the RISC which binds to mRNA transcripts

if perfect match → mRNA is cleaved and degraded

if imperfect match → the RISC binds to the mRNA blocking translation

RNA-induced Transcription Silencing Complex (RITS)

specific siRNAs and miRNAs are transcribed and processed by Dicer and Argonaut and return to the nucleus

in nucleus they form the RITS

RITS binds to an RNA that is being transcribed

RITS then attracts other proteins that modify the chromatin, adding methyl groups and transforming it into heterochromatin to suppress transcription

RNAi in Biotechnology

RNAi studied in the lab

developed as a pharmaceutical agent

therapeutic RNAi attacks diseases caused by overexpression of specific gene or normal expression of abnormal gene product

potential use of RNAi in diagnosis of cancers

RNAi reduces severity of infections by viruses such as HIV, influenza and polio

successful treatment in animal models: viral infection, eye diseases, cancers, inflammatory bowel disease

immunoglobulin system in humans

antigen recognition: immune system binds to foreign substances (antigens)

humoral immunity:production of immunoglobulins (antibodies) that directly bind to antigens

B lymphocytes: B cells → four variable regions that recognize specific antigen, each B cell synthesizes only one type of immunoglobulin

polypeptide chains immunoglobulins

consists of 4 chains

two identical: Light (L) chain and heavy (H) chain form Y-shaped structure

DNA regions organized into L(leader) and V (variable) regions

constant and variable regions

each L and H chain contains constant and variable regions: constant → C-terminus, variable → N-terminus

antibody diversity occurs in part from random recombination of 35-50 different functional LV regions with any different J (joining) regions

40 V, 5 J, and C regions recombine to form a Kappa light chain

51 V, 27 D and 6 J regions to form heavy chain

recombination and hypermutation

mechanism that further increase antibody diversity

recombination event: joins LV region to J region; not precise, results in considerable variation

high hypermutation: random somatic mutation during B cell development introduces more variation into the LVJ region’s sequence

ENCODE

encyclopedia of DNA elements project

goal: identify all functional DNA sequences and determine how elements regulate expression

discovery: more than 80% of human genome contains regulatory elements (once considered “junk DNA”)

Enhancers and promotor elements

promoters elements: 30,000 promoter regions found within protein-coding genes

ENCODE data show enhancer elements influence transcription of several different promoters

enhancer elements: contain promoter elements that initiate transcription