Part:BBa_K1471004

NLS with PolyA.

We had to use this fragments without the use of gel electrophoresis because the small length of the sequences.

NLS

The best understood system for the transport of macromolecules between the cytoplasm and the nucleus is nuclear import pathway. In this pathway, a protein containing a basic nuclear localization signal (NLS) is imported by a heterodimeric import receptor.

This nuclear localization signal or sequence (NLS) is an amino acid sequence that tags a protein for import into the cell nucleus by nuclear transport.Typically, this signal consists of one or more short sequences of positively charged lysines or arginines exposed on the protein surface. Different nuclear localized proteins may share the same NLS.

The active NLS needs to be exposed to the protein surface, the cell has invented mechanisms to expose a hidden or cryptic NLS by protein phosphorylation, dephosphorylation, dissociation of an inhibitory subunit that masks the NLS, processing of a larger precursor, and binding of a hormone to regulate the nuclear import of a protein transcription factor at a certain stage of development, or cell cycle.

It is proposed that a hexapeptide with four arginines and lysines (and histidines, in some cases) is a good candidate for a "core NLS," that acidic domains on proteins to be imported may participate in anchoring them to the transporter cytoplasmic or pore complex NLS-receptor, and that NLS have both a cytoplasmic and a nuclear function.

The interaction between nuclear proteins and transporter proteins in the pore appears to be largely electrostatic and to be disrupted by the binding of mRNA molecules to the same transporter protein, functioning also for the cytoplasmic export of RNA.

Poly-A

The poly-A tail is a long chain of adenine nucleotides that is added to a messenger RNA (mRNA) molecule during RNA processing to increase the stability of the molecule. Immediately after a gene in a eukaryotic cell is transcribed, the new RNA molecule undergoes several modifications known as RNA processing. These modifications alter both ends of the primary RNA transcript to produce a mature mRNA molecule.

The processing of the 3' end adds a poly-A tail to the RNA molecule. First, the 3' end of the transcript is cleaved to free a 3' hydroxyl. Then an enzyme called poly-A polymerase adds a chain of adenine nucleotides to the RNA. This process, called polyadenylation, adds a poly-A tail that is between 100 and 250 residues long. The poly-A tail makes the RNA molecule more stable and prevents its degradation. Additionally, the poly-A tail allows the mature messenger RNA molecule to be exported from the nucleus and translated into a protein by ribosomes in the cytoplasm.

Poly(A) tails enhance the stability and translation of most eukaryotic messenger RNAs, but difficulties in globally measuring poly(A)-tail lengths have impeded greater understanding of poly(A)-tail function. Here we describe poly(A)-tail length profiling by sequencing (PAL-seq) and apply it to measure tail lengths of millions of individual RNAs isolated from yeasts, cell lines, Arabidopsis thaliana leaves, mouse liver, and zebrafish and frog embryos. Poly(A)-tail lengths were conserved between orthologous mRNAs, with mRNAs encoding ribosomal proteins and other ‘housekeeping’ proteins tending to have shorter tails.

The poly(A) tail also protects the mRNA from nucleases, in the course of the life of a mRNA, the poly(A) tail is systematically shortened by poly(A)-specific nucleases. Once the poly(A) tail is too short to allow binding of PAB, the circular structure shown above unravels, and both the cap and mRNA 3’ end become “exposed”.

References:

Classical Nuclear Localization Signals: Definition, Function, and Interaction with Importin α*. (n.d.). Classical Nuclear Localization Signals: Definition, Function, and Interaction with Importin α. Retrieved October 11, 2014, from http://www.jbc.org/content/282/8/5101.full Nuclear localization sequence. (2014, November 10). Wikipedia. Retrieved October 11, 2014, from http://en.wikipedia.org/wiki/Nuclear_localization_sequence Result Filters. (n.d.). National Center for Biotechnology Information. Retrieved October 11, 2014, from http://www.ncbi.nlm.nih.gov/pubmed/8241603 Poly(A)-tail profiling reveals an embryonic switch in translational control. (n.d.). Nature journal. Retrieved October 11, 2014, from http://www.nature.com/nature/journal/v508/n7494/full/nature13007.html The Poly(A) Tail. (n.d.). The RNA Underworld. Retrieved October 12, 2014, from http://aghunt.wordpress.com/2008/06/05/the-polya-tail/ poly-A tail. (n.d.). Nature.com. Retrieved October 12, 2014, from http://www.nature.com/scitable/definition/poly-a-tail-276

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]